Dr. Joy Wu is a board-certified endocrinologist with over 12 years' experience who specializes in treating women and men with osteoporosis and other bone and mineral diseases, including primary hyperparathyroidism, vitamin D deficiency, Paget's disease and fibrous dysplasia. She has a special interest in optimizing skeletal health for those at risk of bone loss from glucocorticoid treatment, cancer therapies, or organ transplant. She works closely with each individual and his/her referring physician to assess fracture risk, and to develop a tailored treatment and monitoring plan.
Dr. Wu directs a broad basic and translational research program that focuses on skeletal development and the bone marrow hematopoietic niche. Her laboratory is currently studying stem cell therapies for bone formation, and the prevention of cancer metastases to bone (joywulab.stanford.edu). She has been honored with awards from the NIH Director’s New Innovator Award, the Endocrine Society, the American Society for Bone and Mineral Research, and the Mary Kay Foundation. Dr. Wu is committed to training the next generation of physician scientists, and serves as Co-Director of the Stanford Internal Medicine Translational Investigator Program.
- Metabolic Bone Disease
- Diabetes and Metabolism
Associate Professor, Medicine - Endocrinology, Gerontology, & Metabolism
Member, Stanford Cancer Institute
Vice Chair, Basic Science, Department of Medicine (2020 - Present)
Associate Member, Stanford Diabetes Research Center (2018 - Present)
Co-Director, Translational Investigator Program, Department of Medicine (2015 - Present)
Honors & Awards
Faculty Women's Forum Allyship Award, Stanford University (2021)
Alford Distinguished Lecture, Baylor College of Medicine (2019)
Cancer Grant Recipient, The Mary Kay Foundation (2013)
NIH Director's New Innovator Award, NIH (2011)
Claflin Distinguished Scholar Award, Massachusetts General Hospital (2009)
Clinical Scientist Program Instructor Development Award, Harvard Stem Cell Institute (2009)
John Haddad Young Investigator Award, Advances in Mineral Metabolism (2008)
Merck Senior Fellow Award, The Endocrine Society (2007)
Endocrine Scholars Award, The Endocrine Society (2006)
Young Investigator Award, American Society for Bone and Mineral Research Annual Meeting (2006)
Alpha Omega Alpha, Duke University School of Medicine (1997)
Marsden Memorial Award in Chemistry, Stanford University (1993)
Phi Beta Kappa, Stanford University (1993)
Boards, Advisory Committees, Professional Organizations
Member, Skeletal Biology Development and Disease Study Section, NIH (2020 - Present)
Basic Science Seat, Board of Directors, The Endocrine Society (2019 - Present)
Leadership Task Force, The Endocrine Society (2015 - 2015)
Board of Directors, Advances in Mineral Metabolism (2013 - 2016)
Membership Enhancement Committee, The American Society for Bone and Mineral Research (2013 - 2016)
Co-Chair, Trainee & Career Development Core Committee, The Endocrine Society (2007 - 2010)
Ex Officio, Council, The Endocrine Society (2006 - 2010)
Member, The American Society for Bone and Mineral Research (2005 - Present)
Member, The Endocrine Society (2003 - Present)
Board Certification: American Board of Internal Medicine, Endocrinology, Diabetes and Metabolism (2006)
Fellowship: Massachusetts General Hospital (2006) MA
Residency: Brigham and Women's Hospital Harvard Medical School (2003) MA
Medical Education: Duke University School of Medicine (2001) NC
MD/PhD, Duke University (2001)
Current Research and Scholarly Interests
Osteoporosis, a disease of fragile bones resulting in fractures, will strike 50% of women and 25% of men. As a physician scientist, my laboratory is studying stem cells in the skeleton and bone marrow to develop novel regenerative approaches to increase bone quality and strength. We are also interested in how the skeleton supports hematopoiesis, and how diseases and medications that impact bone may affect blood cell production and cancer metastasis. For more detailed descriptions of ongoing research projects in the lab, visit our website at joywulab.stanford.edu.
Pilot Trial of Zoledronic Acid to Prevent Bone Loss in Hematopoietic Cell Transplant Recipients
Patients who undergo bone marrow transplant for different types of cancer are exposed to many treatments such as steroids and whole body radiation. These treatments make the transplant possible but also make their bones weaker and more prone to fractures which can be a source of significant disability and decreased quality of life for cancer survivors. Our trial will investigate whether giving one dose of Zoledronic acid (a commonly used drug given to preserve bone mass in osteoporosis) before bone marrow transplant can protect from the bone loss caused by the transplant procedures. The investigators are also interested in studying the complex interactions of bone, muscle and fat which are greatly affected after bone marrow transplant.
Stanford is currently not accepting patients for this trial.
Independent Studies (10)
- Directed Reading in Immunology
IMMUNOL 299 (Win, Spr)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Win, Spr)
- Early Clinical Experience in Medicine
MED 280 (Win, Spr)
- Graduate Research
IMMUNOL 399 (Aut, Win, Spr, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr)
- Medical Scholars Research
MED 370 (Aut, Win, Spr)
- Teaching in Immunology
IMMUNOL 290 (Win, Spr)
- Undergraduate Research
IMMUNOL 199 (Aut, Win, Spr)
- Undergraduate Research
MED 199 (Aut, Win, Spr)
- Directed Reading in Immunology
Doctoral Dissertation Reader (AC)
Graduate and Fellowship Programs
Direct reprogramming of mouse fibroblasts into functional osteoblasts.
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
While induced pluripotent stem cells hold promise as a potential source of osteoblasts for skeletal regeneration, the induction of pluripotency followed by directed differentiation into osteoblasts is time-consuming and low yield. In contrast, direct lineage reprogramming without an intervening stem/progenitor cell stage would be a more efficient approach to generate osteoblasts. We screened combinations of osteogenic transcription factors and identified four factors, Runx2, Osx, Dlx5, and ATF4, that rapidly and efficiently reprogram mouse fibroblasts derived from 2.3 kb type I collagen promoter-driven green fluorescent protein (Col2.3GFP) transgenic mice into induced osteoblast cells (iOBs). iOBs exhibit osteoblast morphology, form mineralized nodules, and express Col2.3GFP and gene markers of osteoblast differentiation. The global transcriptome profiles validated that iOBs resemble primary osteoblasts. Genome-wide DNA methylation analysis demonstrates that within differentially methylated loci, the methylation status of iOBs more closely resembles primary osteoblasts than mouse fibroblasts. We further demonstrate that Col2.3GFP+ iOBs have transcriptome profiles similar to GFP+ cells harvested from Col2.3GFP mouse bone chips. Functionally, Col2.3GFP+ iOBs form mineralized bone structures after subcutaneous implantation in immunodeficient mice and contribute to bone healing in a tibia bone fracture model. These findings provide an approach to derive and study osteoblasts for skeletal regeneration. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/jbmr.3929
View details for PubMedID 31793059
- Pluripotent stem cells as a source of osteoblasts for bone tissue regeneration BIOMATERIALS 2019; 196: 31–45
Older Men with Anemia Have Increased Fracture Risk Independent of Bone Mineral Density.
journal of clinical endocrinology and metabolism
Extreme low hemoglobin values have been linked to increased risk of fracture at different sites in a small number of studies. However, careful assessment of a clinically relevant cutoff for anemia and cross sectional and longitudinal bone mineral density (BMD) measures is lacking.To determine whether men with anemia were at increased risk of fracture after accounting for bone density and bone loss.Cross-sectional analysis (at visit 3) and prospective analysis (from baseline to visit 3) in the Osteoporotic Fractures in Men (MrOS), a multi-site longitudinal cohort study.6 communities in the U.S.3632 community-dwelling men (>65 years) in MrOS at baseline (2000-2002) who were able to walk without assistance and did not have a hip replacement or fracture and had complete blood counts (CBCs) at visit 3 (2007-2009).Adjudicated spine and non-spine fractures during a median 7.2 years follow up.Analytic baseline characteristics associated with fractures or anemia (Hgb < 12g/dL) were included into multivariable models. Anemia was associated with increased risk of any (HR 1.67; 95% CI 1.26-2.21) and non-spine (HR 1.70; 95% CI 1.25-2.31) fractures. A model including change in BMD slightly attenuated the association with any (HR 1.60; 95% CI 1.20-2.13) and non-spine fractures (HR 1.57; 95% CI 1.14-2.15). Including absolute BMD did not significantly alter the anemia-fracture association. Anemia was not associated with spine fracture.Community-dwelling older men with anemia had a 57-72% increase in non-spine fracture risk independent of bone density and bone loss over time.
View details for DOI 10.1210/jc.2017-00266
View details for PubMedID 28368469
Bone Density Loss Is Associated With Blood Cell Counts
JOURNAL OF BONE AND MINERAL RESEARCH
2017; 32 (2): 212-220
Hematopoiesis depends on a supportive microenvironment. Preclinical studies in mice have demonstrated that osteoblasts influence the development of blood cells, particularly erythrocytes, B lymphocytes, and neutrophils. However, it is unknown whether osteoblast numbers or function impact blood cell counts in humans. We tested the hypothesis that men with low BMD or greater BMD loss have decreased circulating erythrocytes and lymphocytes and increased myeloid cells. We performed a cross-sectional analysis and prospective analysis in the Osteoporotic Fractures in Men (MrOS), a multi-site longitudinal cohort study. 2571 community-dwelling men (>65 years) who were able to walk without assistance, did not have a hip replacement or fracture and had complete blood counts (CBCs) at the third study visit were analyzed. Multivariable (MV)-adjusted logistic regression estimated odds of white blood cell subtypes (highest and lowest quintile vs middle), and anemia (clinically defined) associated with BMD by DXA scan (at visit 3), annualized percent BMD change (baseline to visit 3), and high BMD loss (>0.5%/year, from baseline to visit 3) at the femoral neck (FN) and total hip (TH). MV adjusted models included age, BMI, cancer history, smoking status, alcohol intake, corticosteroid use, self-reported health, thiazide use and physical activity. At visit 3 greater TH BMD loss (per standard deviation) was associated with increased odds of anemia, high neutrophils, and low lymphocytes. Annualized BMD loss of >0.5% was associated with increased odds of anemia, high neutrophils, and low lymphocytes. Similar results were observed for FN BMD regarding anemia and lymphocytes. We concluded that community-dwelling older men with declining hip BMD over about 7 years had increased risks of anemia, lower lymphocyte count, and higher neutrophil count, consistent with pre-clinical studies. Bone health and hematopoiesis may have greater interdependency than previously recognized. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/jbmr.3000
View details for Web of Science ID 000396935300004
In vivo rescue of the hematopoietic niche by pluripotent stem cell complementation of defective osteoblast compartments.
Stem cells (Dayton, Ohio)
Bone-forming osteoblasts play critical roles in supporting bone marrow hematopoiesis. Pluripotent stem cells (PSCs), including embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, are capable of differentiating into osteoblasts. To determine the capacity of stem cells needed to rescue aberrant skeletal development and bone marrow hematopoiesis in vivo, we employed a skeletal complementation model. Mice deficient in Runx2, a master transcription factor for osteoblastogenesis, fail to form a mineralized skeleton and bone marrow. Wild-type GFP(+) ES and YFP(+) iPS cells were introduced into Runx2-null blastocyst-stage embryos. We assessed GFP/YFP(+) cell contribution by whole-mount fluorescence and histological analysis and found that the proportion of PSCs in the resulting chimeric embryos is directly correlated with the degree of mineralization in the skull. Moreover, PSC contribution to long bones successfully restored bone marrow hematopoiesis. We validated this finding in a separate model with diphtheria toxin A-mediated ablation of hypertrophic chondrocytes and osteoblasts. Remarkably, chimeric embryos harboring as little as 37.5% wild-type PSCs revealed grossly normal skeletal morphology, suggesting a near-complete rescue of skeletogenesis. In summary, we demonstrate that fractional contribution of PSCs in vivo is sufficient to complement and reconstitute an osteoblast-deficient skeleton and hematopoietic marrow. Further investigation using genetically modified PSCs with conditional loss of gene function in osteoblasts will enable us to address the specific roles of signaling mediators to regulate bone formation and hematopoietic niches in vivo. This article is protected by copyright. All rights reserved.
View details for PubMedID 28741855
Prevention of breast cancer skeletal metastases with parathyroid hormone.
2017; 2 (17)
Advanced breast cancer is frequently associated with skeletal metastases and accelerated bone loss. Recombinant parathyroid hormone [teriparatide, PTH(1-34)] is the first anabolic agent approved in the US for treatment of osteoporosis. While signaling through the PTH receptor in the osteoblast lineage regulates bone marrow hematopoietic niches, the effects of anabolic PTH on the skeletal metastatic niche are unknown. Here, we demonstrate, using orthotopic and intratibial models of 4T1 murine and MDA-MB-231 human breast cancer tumors, that anabolic PTH decreases both tumor engraftment and the incidence of spontaneous skeletal metastasis in mice. Microcomputed tomography and histomorphometric analyses revealed that PTH increases bone volume and reduces tumor engraftment and volume. Transwell migration assays with murine and human breast cancer cells revealed that PTH alters the gene expression profile of the metastatic niche, in particular VCAM-1, to inhibit recruitment of cancer cells. While PTH did not affect growth or migration of the primary tumor, it elicited several changes in the tumor gene expression profile resulting in a less metastatic phenotype. In conclusion, PTH treatment in mice alters the bone microenvironment, resulting in decreased cancer cell engraftment, reduced incidence of metastases, preservation of bone microarchitecture and prolonged survival.
View details for PubMedID 28878134
Loss of Gsa in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy.
journal of biological chemistry
2016; 291 (4): 1631-1642
Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTHR1) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTHR1 exhibited a dramatic increase in trabecular bone that was dependent upon expression of Gsα in the osteoblast lineage. Postnatal removal of Gsα in the osteoblast lineage (P-GsαOsxKO mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1-34) (80 μg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-GsαOsxKO mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-GsαOsxKO mice. In mice that express a mutated PTHR1 that activates adenylyl cyclase and protein kinase A (PKA) via Gsα but not phospholipase C (PLC) via Gq/11 (D/D mice), PTH significantly enhanced bone formation, indicating that PLC activation is not required for increased bone turnover in response to PTH. Therefore while the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of Gsα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond Gsα and Gq/11 act downstream of PTH on osteoblast differentiation.
View details for DOI 10.1074/jbc.M115.679753
View details for PubMedID 26598522
PTH Signaling in Osteoprogenitors Is Essential for B-Lymphocyte Differentiation and Mobilization.
Journal of bone and mineral research
2015; 30 (12): 2273-2286
Cells of the osteoblast lineage provide critical support for B lymphopoiesis in the bone marrow (BM). Parathyroid hormone (PTH) signaling in osteoblastic cells through its receptor (PPR) is an important regulator of hematopoietic stem cells; however, its role in regulation of B lymphopoiesis is not clear. Here we demonstrate that deletion of PPR in osteoprogenitors results in a significant loss of trabecular and cortical bone. PPR signaling in osteoprogenitors, but not in mature osteoblasts or osteocytes, is critical for B-cell precursor differentiation via IL-7 production. Interestingly, despite a severe reduction in B-cell progenitors in BM, mature B-lymphocytes were increased 3.5-fold in the BM of mice lacking PPR in osteoprogenitors. This retention of mature IgD(+) B cells in the BM was associated with increased expression of vascular cell adhesion molecule 1 (VCAM1) by PPR-deficient osteoprogenitors, and treatment with VCAM1 neutralizing antibody increased mobilization of B lymphocytes from mutant BM. Our results demonstrate that PPR signaling in early osteoblasts is necessary for B-cell differentiation via IL-7 secretion and for B-lymphocyte mobilization via VCAM1. © 2015 American Society for Bone and Mineral Research.
View details for DOI 10.1002/jbmr.2581
View details for PubMedID 26191777
Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation.
Proceedings of the National Academy of Sciences of the United States of America
2014; 111 (49): E5262-71
Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated-to our knowledge for the first time-that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation.
View details for DOI 10.1073/pnas.1420463111
View details for PubMedID 25422448
Loss of G(s)alpha Early in the Osteoblast Lineage Favors Adipogenic Differentiation of Mesenchymal Progenitors and Committed Osteoblast Precursors
JOURNAL OF BONE AND MINERAL RESEARCH
2014; 29 (11): 2414-2426
In humans, aging and glucocorticoid treatment are associated with reduced bone mass and increased marrow adiposity, suggesting that the differentiation of osteoblasts and adipocytes may be coordinately regulated. Within the bone marrow, both osteoblasts and adipocytes are derived from mesenchymal progenitor cells, but the mechanisms guiding the commitment of mesenchymal progenitors into osteoblast versus adipocyte lineages are not fully defined. The heterotrimeric G protein subunit Gs α activates protein kinase A signaling downstream of several G protein-coupled receptors including the parathyroid hormone receptor, and plays a crucial role in regulating bone mass. Here we show that targeted ablation of Gs α in early osteoblast precursors, but not in differentiated osteocytes, results in a dramatic increase in bone marrow adipocytes. Mutant mice have reduced numbers of mesenchymal progenitors overall, with an increase in the proportion of progenitors committed to the adipocyte lineage. Furthermore, cells committed to the osteoblast lineage retain adipogenic potential both in vitro and in vivo. These findings have clinical implications for developing therapeutic approaches to direct the commitment of mesenchymal progenitors into the osteoblast lineage. © 2014 American Society for Bone and Mineral Research.
View details for DOI 10.1002/jbmr.2270
View details for Web of Science ID 000344222800011
Teriparatide ( PTH1-34) Treatment Increases Peripheral Hematopoietic Stem Cells in Postmenopausal Women
JOURNAL OF BONE AND MINERAL RESEARCH
2014; 29 (6): 1380-1386
Cells of the osteoblast lineage play an important role in regulating the hematopoietic stem cell (HSC) niche and early B cell development in animal models, perhaps via parathyroid hormone (PTH) dependent mechanisms. There are few human clinical studies investigating this phenomenon. We studied the impact of long-term daily teriparatide (PTH 1-34) treatment on cells of the hematopoietic lineage in postmenopausal women. Twenty-three postmenopausal women at high risk of fracture received teriparatide 20 mcg SC daily for 24 months as part of a prospective longitudinal trial. Whole blood measurements were obtained at baseline, 3, 6, 12, and 18 months. Flow cytometry was performed to identify hematopoietic subpopulations, including HSCs (CD34 + /CD45(moderate); ISHAGE protocol) and early transitional B cells (CD19 + , CD27-, IgD + , CD24[hi], CD38[hi]), CD38[hi]). Serial measurements of spine and hip bone mineral density as well as serum P1NP, osteocalcin, and CTX were also performed. The average age of study subjects was 64 ± 5. We found that teriparatide treatment led to an early increase in circulating HSC number of 40% ± 14% (p = 0.004) by month 3, which persisted to month 18 before returning to near baseline by 24 months. There were no significant changes in transitional B cells or total B cells over the course of the study period. In addition, there were no differences in complete blood count profiles as quantified by standard automated flow cytometry. Interestingly, the peak increase in HSC number was inversely associated with increases in bone markers and spine BMD. Daily teriparatide treatment for osteoporosis increases circulating HSCs by 3 to 6 months in postmenopausal women. This may represent a proliferation of marrow HSCs or increased peripheral HSC mobilization. This clinical study establishes the importance of PTH in the regulation of the HSC niche within humans. © 2014 American Society for Bone and Mineral Research.
View details for DOI 10.1002/jbmr.2171
View details for Web of Science ID 000336001500010
View details for PubMedID 24420643
Histopathology of osteogenesis imperfecta bone. Supramolecular assessment of cells and matrices in the context of woven and lamellar bone formation using light, polarization and ultrastructural microscopy.
2021; 14: 100734
Diaphyseal long bone cortical tissue from 30 patients with lethal perinatal Sillence II and progressively deforming Sillence III osteogenesis imperfecta (OI) has been studied at multiple levels of structural resolution. Interpretation in the context of woven to lamellar bone formation by mesenchymal osteoblasts (MOBLs) and surface osteoblasts (SOBLs) respectively demonstrates lamellar on woven bone synthesis as an obligate self-assembly mechanism and bone synthesis following the normal developmental pattern but showing variable delay in maturation caused by structurally abnormal or insufficient amounts of collagen matrix. The more severe the variant of OI is, the greater the persistence of woven bone and the more immature the structural pattern; the pattern shifts to a structurally stronger lamellar arrangement once a threshold accumulation for an adequate scaffold of woven bone has been reached. Woven bone alone characterizes lethal perinatal variants; variable amounts of woven and lamellar bone occur in progressively deforming variants; and lamellar bone increasingly forms rudimentary and then partially compacted osteons not reaching full compaction. At differing levels of microscopic resolution: lamellar bone is characterized by short, obliquely oriented lamellae with a mosaic appearance in progressively deforming forms; polarization defines tissue conformations and localizes initiation of lamellar formation; ultrastructure of bone forming cells shows markedly dilated rough endoplasmic reticulum (RER) and prominent Golgi bodies with disorganized cisternae and swollen dispersed tubules and vesicles, structural indications of storage disorder/stress responses and mitochondrial swelling in cells with massively dilated RER indicating apoptosis; ultrastructural matrix assessments in woven bone show randomly oriented individual fibrils but also short pericellular bundles of parallel oriented fibrils positioned obliquely and oriented randomly to one another and in lamellar bone show unidirectional fibrils that deviate at slight angles to adjacent bundles and obliquely oriented fibril groups consistent with twisted plywood fibril organization. Histomorphometric indices, designed specifically to document woven and lamellar conformations in normal and OI bone, establish ratios for: i) cell area/total area X 100 indicating the percentage of an area occupied by cells (cellularity index) and ii) total area/number of cells (pericellular matrix domains). Woven bone is more cellular than lamellar bone and OI bone is more cellular than normal bone, but these findings occur in a highly specific fashion with values (high to low) encompassing OI woven, normal woven, OI lamellar and normal lamellar conformations. Conversely, for the total area/number of cells ratio, pericellular matrix accumulations in OI woven are smallest and normal lamellar largest. Since genotype-phenotype correlation is not definitive, interposing histologic/structural analysis allowing for a genotype-histopathologic-phenotype correlation will greatly enhance understanding and clinical management of OI.
View details for DOI 10.1016/j.bonr.2020.100734
View details for PubMedID 33665234
The characterization of distinct populations of murine skeletal cells that have different roles in B lymphopoiesis.
Hematopoiesis is extrinsically controlled by cells of the bone marrow microenvironment, including skeletal lineage cells. The identification and subsequent studies of distinct subpopulations of maturing skeletal cells is currently limited due to a lack of methods to isolate these cells. We found that murine Lineage-CD31-Sca-1-CD51+ cells can be divided into four subpopulations using flow cytometry, based on their expression of the platelet derived growth factor receptors ⍺ and β (PDGFR⍺ and PDGFRβ). The use of different skeletal lineage reporters confirmed the skeletal origin of the four populations. Multiplex immunohistochemistry studies revealed that all four populations were localized near the growth plate and trabecular bone and were rarely found near cortical bone regions or in central bone marrow. Functional studies revealed differences in their abundance, colony-forming unit-fibroblast capacity and potential to differentiate into mineralized osteoblasts or adipocytes in vitro. Furthermore, the four populations had distinct gene expression profiles and differential cell surface expression of leptin receptor (LEPR) and vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we discovered that one of these four different skeletal populations showed the highest expression of genes involved in the extrinsic regulation of B lymphopoiesis. This cell population varied in abundance between distinct hematopoietically active skeletal sites, and significant differences in the proportions of B lymphocyte precursors were also observed in these distinct skeletal sites. It also supported pre-B lymphopoiesis in culture. Our method to isolate four distinct maturing skeletal populations will assist in elucidating the roles of distinct skeletal niche cells in regulating hematopoiesis and bone.
View details for DOI 10.1182/blood.2020005865
View details for PubMedID 33786586
- Finding My Voice. Annals of internal medicine 2021
- Increased NF-kappa B Activity in Osteoprogenitor-Lineage Cells Impairs the Balance of Bone Versus Fat in the Marrow of Skeletally Mature Mice REGENERATIVE ENGINEERING AND TRANSLATIONAL MEDICINE 2020; 6 (1): 69–77
Induction of Osteoblasts by Direct Reprogramming of Mouse Fibroblasts.
Methods in molecular biology (Clifton, N.J.)
2020; 2155: 201–12
In the tissue culture dish, osteoblast cells can be derived from mesenchymal stem cells (MSCs) and pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, differentiation of osteoblasts from PSCs is time-consuming and low yield. In contrast, we identified four osteogenic transcription factors, Runx2, Osx, Dlx5, and ATF4, that rapidly and efficiently reprogram mouse fibroblasts derived from 2.3 kb type I collagen promoter-driven green fluorescent protein (Col2.3GFP) transgenic mice into induced osteoblast cells (iOBs). iOBs exhibit osteoblast morphology, form mineralized nodules, and express Col2.3GFP and gene markers of osteoblast differentiation. Our method provides a robust system to rapidly generate appropriate and abundant osteoblast cells for osteogenesis and bone regeneration study.
View details for DOI 10.1007/978-1-0716-0655-1_17
View details for PubMedID 32474879
Calcinosis is associated with ischemic manifestations and increased disability in patients with systemic sclerosis.
Seminars in arthritis and rheumatism
2020; 50 (5): 891–96
Calcinosis is a debilitating complication of systemic sclerosis (SSc) with no effective treatments. We sought to identify clinical correlations and to characterize complications and disability associated with calcinosis in a multi-center, international cohort of SSc patients.We established a cohort of 568 consecutive SSc patients who fulfill 2013 revised ACR/EULAR criteria at 10 centers within North America, Australia, and Mexico. Calcinosis was defined as subcutaneous calcium deposition by imaging and/or physical examination, or a clear history of extruded calcium. All patients completed the Scleroderma Health Assessment Questionnaire Disability Index and Cochin Hand Functional Scale.215 (38%) patients had calcinosis. In multivariable analysis, disease duration (OR=1.24, p = 0.029), digital ischemia (OR=1.8, p = 0.002) and Acro-osteolysis (OR=2.97, p = 0.008) were significantly associated with calcinosis. In the subset of patients with bone densitometry (n = 68), patients with calcinosis had significantly lower median T-scores than patients without (-2.2 vs. -1.7, p = 0.004). The most common location of calcinosis lesions was the hands (70%), particularly the thumbs (19%) with decreasing frequency moving to the fifth fingers (8%). The most common complications were tenderness (29% of patients) and spontaneous extrusion of calcinosis through the skin (20%), while infection was rare (2%). Disability and hand function were worse in patients with calcinosis, particularly if locations in addition to the fingers/thumbs were involved.We confirmed a strong association between calcinosis and digital ischemia. Calcinosis in SSc patients most commonly affects the hands and is associated with a high burden of disability and hand dysfunction.
View details for DOI 10.1016/j.semarthrit.2020.06.007
View details for PubMedID 32898758
Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes.
Cell stem cell
2020; 27 (1): 50–63.e5
Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3β inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.
View details for DOI 10.1016/j.stem.2020.06.001
View details for PubMedID 32619518
Bone Health Management After Hematopoietic Cell Transplantation: An Expert Panel Opinion from the American Society for Transplantation and Cellular Therapy.
Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation
Bone health disturbances occur commonly after hematopoietic cell transplantation (HCT) with loss of bone mineral density (BMD) and avascular necrosis (AVN) being foremost. BMD loss is related to pre-transplant chemotherapy and radiation exposures, immunosuppressive therapy for graft-versus-host-disease (GVHD), and results from deficiencies in growth or gonadal hormones, disturbances in calcium and vitamin D homeostasis, as well as osteoblast and osteoclast dysfunction. While the pathophysiology of AVN remains unclear, high-dose glucocorticoid exposure is the most frequent association. Different societal treatment guidelines for osteoporosis exist but focus mainly on menopausal-associated osteoporosis. HCT survivors comprise a distinct population with unique comorbidities, making general approaches to bone health management sometimes inappropriate. To address a core set of 16 frequently asked questions (FAQ) relevant to bone health in HCT, the American Society of Transplant and Cellular Therapy (ASTCT) Committee on Practice Guidelines convened a panel of experts in HCT, adult and pediatric endocrinology, orthopedics and oral medicine. Due to a lack of relevant prospective controlled clinical trials that specifically address bone health in HCT, the answers to the presented FAQs rely on evidence derived from retrospective HCT studies, results extrapolated from prospective studies in non-HCT settings, relevant societal guidelines, and expert panel opinion. Given heterogenous comorbidities and needs of individual HCT recipients, answers to FAQs in this article should be considered as general recommendations with good medical practice and judgment ultimately dictating care of individual patients. Readers are referred to the supplement for answers to additional FAQs that did not make the core set.
View details for DOI 10.1016/j.bbmt.2020.07.001
View details for PubMedID 32653624
- Preconditioned or IL4-Secreting Mesenchymal Stem Cells Enhanced Osteogenesis at Different Stages TISSUE ENGINEERING PART A 2019
- Association of bone mineral density with hemoglobin and change in hemoglobin among older men and women: The Cardiovascular Health Study BONE 2019; 120: 321–26
- Mesenchymal lineage cells and their importance in B lymphocyte niches BONE 2019; 119: 42–56
Woven bone overview: structural classification based on its integral role in developmental, repair and pathological bone formation throughout vertebrate groups.
European cells & materials
2019; 38: 137–67
Cortical bone development is characterised by initial formation of woven bone followed by deposition of lamellar bone on the woven scaffold. This occurs in normal bone formation as an integral obligate self-assembly pattern throughout all vertebrate groups, with specific temporal and spatial features. It also occurs in repair bone, modified by the biophysical/mechanical environment, and in pathological bone, modified by the specific disorder and its severity. Two spatially distinct osteoblast cell populations synthesise woven and lamellar bone: mesenchymal osteoblasts surround themselves circumferentially with collagen in a random array to form woven bone; surface osteoblasts align themselves in a linear array on the woven bone surface (or adjacent lamellar bone) to synthesise parallel-fibred lamellar bone. Four specific stages of woven bone formation are defined: stage I, early differentiation of pre-osteoblasts from undifferentiated mesenchymal cells; stage II, mesenchymal osteoblasts surrounding themselves in a 360° arc with randomly oriented matrix fibres; stage III, woven matrix acting as a scaffold on which surface osteoblasts begin to synthesise bone in parallel-fibred lamellar conformation; stage IV, progressive relative diminution of woven bone in the woven bone/lamellar bone complex. Stages II and IV are further subdivided (in a, b and c) by shifting cell area/matrix area and woven bone/lamellar bone relationships. The under-appreciated biological significance of woven bone is that it initiates formation de novo at sites of no previous bone. This information allows for targeted assessment of molecular-biophysical mechanisms underlying woven bone formation and their utilisation for initiating enhanced bone formation.
View details for DOI 10.22203/eCM.v038a11
View details for PubMedID 31571191
Constitutive stimulatory G protein activity in limb mesenchyme impairs bone growth.
2018; 110: 230–37
GNAS mutations leading to constitutively active stimulatory G protein alpha-subunit (Gsα) cause different tumors, fibrous dysplasia of bone, and McCune-Albright syndrome, which are typically not associated with short stature. Enhanced signaling of the parathyroid hormone/parathyroid hormone-related peptide receptor, which couples to multiple G proteins including Gsα, leads to short bones with delayed endochondral ossification. It has remained unknown whether constitutive Gsα activity also impairs bone growth. Here we generated mice expressing a constitutively active Gsα mutant (Gsα-R201H) conditionally upon Cre recombinase (cGsαR201Hmice). Gsα-R201H was expressed in cultured bone marrow stromal cells from cGsαR201Hmice upon adenoviral-Cre transduction. When crossed with mice in which Cre is expressed in a tamoxifen-regulatable fashion (CAGGCre-ER™), tamoxifen injection resulted in mosaic expression of the transgene in double mutant offspring. We then crossed the cGsαR201Hmice with Prx1-Cre mice, in which Cre is expressed in early limb-bud mesenchyme. The double mutant offspring displayed short limbs at birth, with narrow hypertrophic chondrocyte zones in growth plates and delayed formation of secondary ossification center. Consistent with enhanced Gsα signaling, bone marrow stromal cells from these mice demonstrated increased levels of c-fos mRNA. Our findings indicate that constitutive Gsα activity during limb development disrupts endochondral ossification and bone growth. Given that Gsα haploinsufficiency also leads to short bones, as in patients with Albright's hereditary osteodystrophy, these results suggest that a tight control of Gsα activity is essential for normal growth plate physiology.
View details for PubMedID 29471062
Bone and blood interactions in human health and disease.
Under physiologic conditions hematopoiesis takes place in the bone marrow, and the skeleton provides the structural and supportive network necessary for normal hematopoiesis. Chronic disorders affecting hematopoiesis such as sickle cell anemia and thalassemia demonstrate striking skeletal phenotypes including bone loss and increased fracture risk. There is mounting evidence that anemia in older populations may also be associated with bone fragility. Given the interconnectedness of bone and hematopoietic cells, it is important to review the potential clinical implications and opportunities for therapeutic intervention. There are recognized associations between blood-borne and solid tissue malignancy and skeletal health, but our review will focus on non-malignant disease.
View details for PubMedID 29476979
Foxp3(+) regulatory T cells maintain the bone marrow microenvironment for B cell lymphopoiesis
Foxp3(+) regulatory T cells (Treg cells) modulate the immune system and maintain self-tolerance, but whether they affect haematopoiesis or haematopoietic stem cell (HSC)-mediated reconstitution after transplantation is unclear. Here we show that B-cell lymphopoiesis is impaired in Treg-depleted mice, yet this reduced B-cell lymphopoiesis is rescued by adoptive transfer of affected HSCs or bone marrow cells into Treg-competent recipients. B-cell reconstitution is abrogated in both syngeneic and allogeneic transplantation using Treg-depleted mice as recipients. Treg cells can control physiological IL-7 production that is indispensable for normal B-cell lymphopoiesis and is mainly sustained by a subpopulation of ICAM1(+) perivascular stromal cells. Our study demonstrates that Treg cells are important for B-cell differentiation from HSCs by maintaining immunological homoeostasis in the bone marrow microenvironment, both in physiological conditions and after bone marrow transplantation.
View details for DOI 10.1038/ncomms15068
View details for PubMedID 28485401
Gs alpha Controls Cortical Bone Quality by Regulating Osteoclast Differentiation via cAMP/PKA and beta-Catenin Pathways
Skeletal bone formation and maintenance requires coordinate functions of several cell types, including bone forming osteoblasts and bone resorbing osteoclasts. Gsα, the stimulatory subunit of heterotrimeric G proteins, activates downstream signaling through cAMP and plays important roles in skeletal development by regulating osteoblast differentiation. Here, we demonstrate that Gsα signaling also regulates osteoclast differentiation during bone modeling and remodeling. Gnas, the gene encoding Gsα, is imprinted. Mice with paternal allele deletion of Gnas (Gnas(+/p-)) have defects in cortical bone quality and strength during early development (bone modeling) that persist during adult bone remodeling. Reduced bone quality in Gnas(+/p-) mice was associated with increased endosteal osteoclast numbers, with no significant effects on osteoblast number and function. Osteoclast differentiation and resorption activity was enhanced in Gnas(+/p-) cells. During differentiation, Gnas(+/p-) cells showed diminished pCREB, β-catenin and cyclin D1, and enhanced Nfatc1 levels, conditions favoring osteoclastogenesis. Forskolin treatment increased pCREB and rescued osteoclast differentiation in Gnas(+/p-) by reducing Nfatc1 levels. Cortical bone of Gnas(+/p-) mice showed elevated expression of Wnt inhibitors sclerostin and Sfrp4 consistent with reduced Wnt/β-catenin signaling. Our data identify a new role for Gsα signaling in maintaining bone quality by regulating osteoclast differentiation and function through cAMP/PKA and Wnt/β-catenin pathways.
View details for DOI 10.1038/srep45140
View details for PubMedID 28338087
Parathyroid Hormone Directs Bone Marrow Mesenchymal Cell Fate.
Intermittent PTH administration builds bone mass and prevents fractures, but its mechanism of action is unclear. We genetically deleted the PTH/PTHrP receptor (PTH1R) in mesenchymal stem cells using Prx1Cre and found low bone formation, increased bone resorption, and high bone marrow adipose tissue (BMAT). Bone marrow adipocytes traced to Prx1 and expressed classic adipogenic markers and high receptor activator of nuclear factor kappa B ligand (Rankl) expression. RANKL levels were also elevated in bone marrow supernatant and serum, but undetectable in other adipose depots. By cell sorting, Pref1(+)RANKL(+) marrow progenitors were twice as great in mutant versus control marrow. Intermittent PTH administration to control mice reduced BMAT significantly. A similar finding was noted in male osteoporotic patients. Thus, marrow adipocytes exhibit osteogenic and adipogenic characteristics, are uniquely responsive to PTH, and secrete RANKL. These studies reveal an important mechanism for PTH's therapeutic action through its ability to direct mesenchymal cell fate.
View details for DOI 10.1016/j.cmet.2017.01.001
View details for PubMedID 28162969
Evidence for use of Teriparatide in Spinal Fusion Surgery in Osteoporotic Patients.
Osteoporosis is defined as a bone mineral density (BMD) less than 2.5 standard deviations below the mean BMD at peak bone mass, or the presence of a fragility fracture. In the setting of osteoporosis, early hardware loosening is thought to cause decreased spinal fusion rates. The two mainstays of osteoporosis treatment are bisphosphonates and Teriparatide. Teriparatide, a form of synthetic parathyroid hormone (PTH), is an anabolic agent that increases osteoblast activity and, thereby, bone mass. Preclinical studies in animal models show that Teriparatide increases spinal fusion rates. Early clinical studies show that teriparatide both increases spinal fusion rates and decreases hardware loosening in the setting of postmenopausal osteoporosis. Ongoing additional trials will help formulate preoperative screening recommendations, determine the optimal duration of pre- and post-operative Teriparatide treatment, and investigate its utility in men.
View details for DOI 10.1016/j.wneu.2016.11.135
View details for PubMedID 27923758
Calcinosis is associated with digital ulcers and osteoporosis in patients with systemic sclerosis: A Scleroderma Clinical Trials Consortium study
SEMINARS IN ARTHRITIS AND RHEUMATISM
2016; 46 (3): 344-349
We sought to identify the clinical factors associated with calcinosis in an international multicenter collaborative effort with the Scleroderma Clinical Trials Consortium (SCTC).This is a retrospective cohort study of 5218 patients with systemic sclerosis (SSc). Logistic regression was used to obtain odds ratios (OR) relating calcinosis to various clinical features in multivariate analyses.A total of 1290 patients (24.7%) had calcinosis. In univariate analyses, patients with calcinosis were older than patients without calcinosis, more likely to be female, and had longer disease duration from the first non-Raynaud phenomenon symptom. Patients with calcinosis were more likely to have digital ulcers, telangiectasias, acro-osteolysis, cardiac disease, pulmonary hypertension, gastrointestinal involvement, arthritis, and osteoporosis, but less likely to have muscle disease. Anti-Scl-70, RNA-polymerase-III, and U1-RNP autoantibodies were significantly less common in patients with calcinosis, while anticentromere (ACA), anti-PM/Scl, and anticardiolipin antibodies were more frequent. In multivariate analysis, the strongest associations with calcinosis were digital ulcers (OR = 3.9; 95% CI: 2.7-5.5; p < 0.0001) and osteoporosis (OR = 4.2; 95% CI: 2.3-7.9; p < 0.0001).One quarter of patients with SSc have calcinosis at some time during their illness. Our data confirm a strong association of calcinosis with digital ulcers, and support a novel association with osteoporosis.
View details for DOI 10.1016/j.semarthrit.2016.05.008
View details for Web of Science ID 000390979200012
View details for PubMedID 27371996
SIKs control osteocyte responses to parathyroid hormone
Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.
View details for DOI 10.1038/ncomms13176
View details for PubMedID 27759007
Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the, bone marrow
NATURE CELL BIOLOGY
2016; 18 (10): 1078-1089
Breast cancer cells frequently home to the bone marrow, where they may enter a dormant state before forming a bone metastasis. Several members of the interleukin-6 (IL-6) cytokine family are implicated in breast cancer bone colonization, but the role for the IL-6 cytokine leukaemia inhibitory factor (LIF) in this process is unknown. We tested the hypothesis that LIF provides a pro-dormancy signal to breast cancer cells in the bone. In breast cancer patients, LIF receptor (LIFR) levels are lower with bone metastases and are significantly and inversely correlated with patient outcome and hypoxia gene activity. Hypoxia also reduces the LIFR:STAT3:SOCS3 signalling pathway in breast cancer cells. Loss of the LIFR or STAT3 enables otherwise dormant breast cancer cells to downregulate dormancy-, quiescence- and cancer stem cell-associated genes, and to proliferate in and specifically colonize the bone, suggesting that LIFR:STAT3 signalling confers a dormancy phenotype in breast cancer cells disseminated to bone.
View details for DOI 10.1038/ncb3408
View details for Web of Science ID 000384961700007
View details for PubMedID 27642788
- Bone Marrow Hematopoietic Niches OSTEOIMMUNOLOGY: INTERACTIONS OF THE IMMUNE AND SKELETAL SYSTEMS, 2ND EDITION 2016: 103–19
Pluripotent Stem Cells and Skeletal Regeneration-Promise and Potential
CURRENT OSTEOPOROSIS REPORTS
2015; 13 (5): 342-350
The bone is a regenerative tissue, capable of healing itself after fractures. However, some circumstances such as critical-size defects, malformations, and tumor destruction may exceed the skeleton's capacity for self-repair. In addition, bone mass and strength decline with age, leading to an increase in fragility fractures. Therefore, the ability to generate large numbers of patient-specific osteoblasts would have enormous clinical implications for the treatment of skeletal defects and diseases. This review will highlight recent advances in the derivation of pluripotent stem cells, and in their directed differentiation towards bone-forming osteoblasts.
View details for DOI 10.1007/s11914-015-0285-9
View details for Web of Science ID 000360692900010
RAR gamma is a negative regulator of osteoclastogenesis
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY
2015; 150: 46-53
Vitamin A is known to influence post-natal bone content, with excess intake being associated with reduced bone mineral density and increased fracture risk. Despite this, the roles retinoids play in regulating osteoclastogenesis, particularly in vivo, remain unresolved. This study therefore aimed to determine the effect of loss of retinoic acid receptors (RAR)α or RARγ on bone mass (analyzed by histomorphometry and dual-energy X-ray absorptiometry) and osteoclastogenesis in mice in vivo. RARγ null mice had significantly less trabecular bone at 8 weeks of age compared to wildtype littermates. In contrast, no change in trabecular bone mass was detected in RARα null mice at this age. Further histomorphometric analysis revealed a significantly greater osteoclast surface in bones from 8-week-old RARγ null male mice. This in vivo effect was cell lineage autonomous, and was associated with increased osteoclastogenesis in vitro from hematopoietic cells obtained from 8-week-old RARγ null male mice. The use of highly selective agonists in RANKL-induced osteoclast differentiation of wild type mouse whole bone marrow cells and RAW264.7 cells in vitro showed a stronger inhibitory effect of RARγ than RARα agonists, suggesting that RARγ is a more potent inhibitor of osteoclastogenesis. Furthermore, NFAT activation was also more strongly inhibited by RARγ than RARα agonists. While RARα and RARγ antagonists did not significantly affect osteoclast numbers in vitro, larger osteoclasts were observed in cultures stimulated with the antagonists, suggesting increased osteoclast fusion. Further investigation into the effect of retinoids in vivo revealed that oral administration of 5mg/kg/day ATRA for 10 days protected against bone loss induced by granulocyte colony-stimulating factor (G-CSF) by inhibiting the pro-osteoclastogenic action of G-CSF. Collectively, our data indicates a physiological role for RARγ as a negative regulator of osteoclastogenesis in vivo and in vitro, and reveals distinct influences of RARα and RARγ in bone structure regulation.
View details for DOI 10.1016/j.jsbmb.2015.03.005
View details for Web of Science ID 000355370300006
Activation of the Wnt/Planar Cell Polarity Pathway Is Required for Pericyte Recruitment during Pulmonary Angiogenesis.
American journal of pathology
2015; 185 (1): 69-84
Pericytes are perivascular cells localized to capillaries that promote vessel maturation, and their absence can contribute to vessel loss. Whether impaired endothelial-pericyte interaction contributes to small vessel loss in pulmonary arterial hypertension (PAH) is unclear. Using 3G5-specific, immunoglobulin G-coated magnetic beads, we isolated pericytes from the lungs of healthy subjects and PAH patients, followed by lineage validation. PAH pericytes seeded with healthy pulmonary microvascular endothelial cells failed to associate with endothelial tubes, resulting in smaller vascular networks compared to those with healthy pericytes. After the demonstration of abnormal polarization toward endothelium via live-imaging and wound-healing studies, we screened PAH pericytes for abnormalities in the Wnt/planar cell polarity (PCP) pathway, which has been shown to regulate cell motility and polarity in the pulmonary vasculature. PAH pericytes had reduced expression of frizzled 7 (Fzd7) and cdc42, genes crucial for Wnt/PCP activation. With simultaneous knockdown of Fzd7 and cdc42 in healthy pericytes in vitro and in a murine model of angiogenesis, motility and polarization toward pulmonary microvascular endothelial cells were reduced, whereas with restoration of both genes in PAH pericytes, endothelial-pericyte association was improved, with larger vascular networks. These studies suggest that the motility and polarity of pericytes during pulmonary angiogenesis are regulated by Wnt/PCP activation, which can be targeted to prevent vessel loss in PAH.
View details for DOI 10.1016/j.ajpath.2014.09.013
View details for PubMedID 25447046
Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow.
The Journal of experimental medicine
Production of the cells that ultimately populate the thymus to generate α/β T cells has been controversial, and their molecular drivers remain undefined. Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells. Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function. The Notch ligand DLL4 is abundantly expressed on bone marrow Ocn(+) cells, and selective depletion of DLL4 from these cells recapitulated the thymopoietic abnormality. These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell-based adaptive immunity.
View details for DOI 10.1084/jem.20141843
View details for PubMedID 25918341
The PTH-G alpha(s)-Protein Kinase A Cascade Controls alpha NAC Localization To Regulate Bone Mass
MOLECULAR AND CELLULAR BIOLOGY
2014; 34 (9): 1622-1633
The binding of PTH to its receptor induces Gαs-dependent cAMP accumulation to turn on effector kinases, including protein kinase A (PKA). The phenotype of mice with osteoblasts specifically deficient for Gαs is mimicked by a mutation leading to cytoplasmic retention of the transcriptional coregulator αNAC, suggesting that Gαs and αNAC form part of a common genetic pathway. We show that treatment of osteoblasts with PTH(1-34) or the PKA-selective activator 6Bnz-cAMP lead to translocation of αNAC to the nucleus. αNAC was phosphorylated by PKA at serine 99 in vitro. Phospho-S99-αNAC accumulated in osteoblasts exposed to PTH(1-34) or 6Bnz-cAMP but not in treated cells expressing dominant negative PKA. Nuclear accumulation was abrogated by an S99A mutation but enhanced by a phosphomimetic residue (S99D). ChIP analysis showed that PTH(1-34) or 6Bnz-cAMP treatment leads to accumulation of αNAC at the Osteocalcin (Ocn) promoter. Altered gene dosage for Gαs and αNAC in compound heterozygous mice results in reduced bone mass, increased numbers of osteocytes, and enhanced expression of Sost. Our results show that αNAC is a substrate of PKA following PTH signaling. This enhances αNAC translocation to the nucleus and leads to its accumulation at target promoters to regulate transcription and affect bone mass.
View details for DOI 10.1128/MCB.01434-13
View details for Web of Science ID 000334316300007
View details for PubMedID 24550008
Mesenchymal progenitors and the osteoblast lineage in bone marrow hematopoietic niches.
Current osteoporosis reports
2014; 12 (1): 22-32
The bone marrow cavity is essential for the proper development of the hematopoietic system. In the last few decades, it has become clear that mesenchymal stem/progenitor cells as well as cells of the osteoblast lineage, besides maintaining bone homeostasis, are also fundamental regulators of bone marrow hematopoiesis. Several studies have demonstrated the direct involvement of mesenchymal and osteoblast lineage cells in the maintenance and regulation of supportive microenvironments necessary for quiescence, self-renewal and differentiation of hematopoietic stem cells. In addition, specific niches have also been identified within the bone marrow for maturing hematopoietic cells. Here we will review recent findings that have highlighted the roles of mesenchymal progenitors and cells of the osteoblast lineage in regulating distinct stages of hematopoiesis.
View details for DOI 10.1007/s11914-014-0190-7
View details for PubMedID 24477415
Differential regulation of myeloid leukemias by the bone marrow microenvironment.
2013; 19 (11): 1513-1517
Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM) and may be the cause of relapse following chemotherapy. Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. CD44 (refs. 3,4) and interleukin-6 (ref. 5) have been implicated previously in the LSC niche. Transforming growth factor-β1 (TGF-β1) is released during bone remodeling and plays a part in maintenance of CML LSCs, but a role for TGF-β1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell-specific activation of the parathyroid hormone (PTH) receptor attenuates BCR-ABL1 oncogene-induced CML-like myeloproliferative neoplasia (MPN) but enhances MLL-AF9 oncogene-induced AML in mouse transplantation models, possibly through opposing effects of increased TGF-β1 on the respective LSCs. PTH treatment caused a 15-fold decrease in LSCs in wild-type mice with CML-like MPN and reduced engraftment of immune-deficient mice with primary human CML cells. These results demonstrate that LSC niches in CML and AML are distinct and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSCs, a prerequisite for the cure of CML.
View details for DOI 10.1038/nm.3364
View details for PubMedID 24162813
Interactions between B lymphocytes and the osteoblast lineage in bone marrow.
Calcified tissue international
2013; 93 (3): 261-268
The regulatory effects of the immune system on the skeleton during homeostasis and activation have been appreciated for years. In the past decade it has become evident that bone tissue can also regulate immune cell development. In the bone marrow, the differentiation of hematopoietic progenitors requires specific microenvironments, called "niches," provided by various subsets of stromal cells, many of which are of mesenchymal origin. Among these stromal cell populations, cells of the osteoblast lineage serve a supportive function in the maintenance of normal hematopoiesis, and B lymphopoiesis in particular. Within the osteoblast lineage, distinct differentiation stages exert differential regulatory effects on hematopoietic development. In this review we will highlight the critical role of osteoblast progenitors in the perivascular B lymphocyte niche.
View details for DOI 10.1007/s00223-013-9753-3
View details for PubMedID 23839529
Myelopoiesis is regulated by osteocytes through Gsa-dependent signaling.
2013; 121 (6): 930-939
Hematopoietic progenitors are regulated in their respective niches by cells of the bone marrow microenvironment. The bone marrow microenvironment is composed of a variety of cell types, and the relative contribution of each of these cells for hematopoietic lineage maintenance has remained largely unclear. Osteocytes, the most abundant yet least understood cells in bone, are thought to initiate adaptive bone remodeling responses via osteoblasts and osteoclasts. Here we report that these cells regulate hematopoiesis, constraining myelopoiesis through a Gsα-mediated mechanism that affects G-CSF production. Mice lacking Gsα in osteocytes showed a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. This hematopoietic phenomenon was neither intrinsic to the hematopoietic cells nor dependent on osteoblasts but was a consequence of an altered bone marrow microenvironment imposed by Gsα deficiency in osteocytes. Conditioned media from osteocyte-enriched bone explants significantly increased myeloid colony formation in vitro, which was blocked by G-CSF–neutralizing antibody, indicating a critical role of osteocyte-derived G-CSF in the myeloid expansion.
View details for DOI 10.1182/blood-2012-06-437160
View details for PubMedID 23160461
- Development of the skeleton Osteoporosis, 4th Edition Academic Press. 2013: 97–126
G(s)alpha enhances commitment of mesenchymal progenitors to the osteoblast lineage but restrains osteoblast differentiation in mice
JOURNAL OF CLINICAL INVESTIGATION
2011; 121 (9): 3492-3504
The heterotrimeric G protein subunit Gsα stimulates cAMP-dependent signaling downstream of G protein-coupled receptors. In this study, we set out to determine the role of Gsα signaling in cells of the early osteoblast lineage in vivo by conditionally deleting Gsα from osterix-expressing cells. This led to severe osteoporosis with fractures at birth, a phenotype that was found to be the consequence of impaired bone formation rather than increased resorption. Osteoblast number was markedly decreased and osteogenic differentiation was accelerated, resulting in the formation of woven bone. Rapid differentiation of mature osteoblasts into matrix-embedded osteocytes likely contributed to depletion of the osteoblast pool. In addition, the number of committed osteoblast progenitors was diminished in both bone marrow stromal cells (BMSCs) and calvarial cells of mutant mice. In the absence of Gsα, expression of sclerostin and dickkopf1 (Dkk1), inhibitors of canonical Wnt signaling, was markedly increased; this was accompanied by reduced Wnt signaling in the osteoblast lineage. In summary, we have shown that Gsα regulates bone formation by at least two distinct mechanisms: facilitating the commitment of mesenchymal progenitors to the osteoblast lineage in association with enhanced Wnt signaling; and restraining the differentiation of committed osteoblasts to enable production of bone of optimal mass, quality, and strength.
View details for DOI 10.1172/JCI46406
View details for Web of Science ID 000294753700017
View details for PubMedID 21804192
Potent constitutive cyclic AMP-generating activity of XL alpha s implicates this imprinted GNAS product in the pathogenesis of McCune-Albright Syndrome and fibrous dysplasia of bone
2011; 48 (2): 312-320
Patients with McCune-Albright syndrome (MAS), characterized primarily by hyperpigmented skin lesions, precocious puberty, and fibrous dyslasia of bone, carry postzygotic heterozygous mutations of GNAS causing constitutive cAMP signaling. GNAS encodes the α-subunit of the stimulatory G protein (Gsα), as well as a large variant (XLαs) derived from the paternal allele. The mutations causing MAS affect both GNAS products, but whether XLαs, like Gsα, can be involved in the pathogenesis remains unknown. Here, we investigated biopsy samples from four previously reported and eight new patients with MAS. Activating mutations of GNAS (Arg201 with respect to the amino acid sequence of Gsα) were present in all the previously reported and five of the new cases. The mutation was detected within the paternally expressed XLαs transcript in five and the maternally expressed NESP55 transcript in four cases. Tissues carrying paternal mutations appeared to have higher XLαs mRNA levels than maternal mutations. The human XLαs mutant analogous to Gsα-R201H (XLαs-R543H) showed markedly higher basal cAMP accumulation than wild-type XLαs in transfected cells. Wild-type XLαs demonstrated higher basal and isoproterenol-induced cAMP signaling than Gsα and co-purified with Gβ1γ2 in transduced cells. XLαs mRNA was measurable in mouse calvarial cells, with its level being significantly higher in undifferentiated cells than those expressing preosteoblastic markers osterix and alkaline phosphatase. XLαs mRNA was also expressed in murine bone marrow stromal cells and preosteoblastic MC3T3-E1 cells. Our findings are consistent with the possibility that constitutive XLαs activity adds to the molecular pathogenesis of MAS and fibrous dysplasia of bone.
View details for DOI 10.1016/j.bone.2010.09.032
View details for Web of Science ID 000286543700019
View details for PubMedID 20887824
- The role of bone cells in establishing the hematopoietic stem cell niche Osteoimmunology: Interactions of the Immune and Skeletal Systems 2011: 81–99
- Clarifying the contributions of distinct mesenchymal populations in supporting hematopoiesis [editorial] IBMS BoneKEy 2010; 7: 369-372
- Role of the Osteoblast Lineage in the Bone Marrow Hematopoietic Niches JOURNAL OF BONE AND MINERAL RESEARCH 2009; 24 (5): 759-764
Osteoblastic regulation of B lymphopoiesis is mediated by G(s)alpha-dependent signaling pathways
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2008; 105 (44): 16976-16981
Osteoblasts play an increasingly recognized role in supporting hematopoietic development and recently have been implicated in the regulation of B lymphopoiesis. Here we demonstrate that the heterotrimeric G protein alpha subunit G(s)alpha is required in cells of the osteoblast lineage for normal postnatal B lymphocyte production. Deletion of G(s)alpha early in the osteoblast lineage results in a 59% decrease in the percentage of B cell precursors in the bone marrow. Analysis of peripheral blood from mutant mice revealed a 67% decrease in the number of circulating B lymphocytes by 10 days of age. Strikingly, other mature hematopoietic lineages are not decreased significantly. Mice lacking G(s)alpha in cells of the osteoblast lineage exhibit a reduction in pro-B and pre-B cells. Furthermore, interleukin (IL)-7 expression is attenuated in G(s)alpha-deficient osteoblasts, and exogenous IL-7 is able to restore B cell precursor populations in the bone marrow of mutant mice. Finally, the defect in B lymphopoiesis can be rescued by transplantation into a WT microenvironment. These findings confirm that osteoblasts are an important component of the B lymphocyte niche and demonstrate in vivo that G(s)alpha-dependent signaling pathways in cells of the osteoblast lineage extrinsically regulate bone marrow B lymphopoiesis, at least partially in an IL-7-dependent manner.
View details for DOI 10.1073/pnas.0802898105
View details for Web of Science ID 000260913800034
View details for PubMedID 18957542
- Development of the Skeleton Osteoporosis, 3rd Edition Academic Press. 2008: 241–269
Spermatogenesis and the regulation of Ca2+-calmodulin-dependent protein kinase IV localization are not dependent on calspermin
MOLECULAR AND CELLULAR BIOLOGY
2001; 21 (17): 6066-6070
Calspermin and Ca(2+)-calmodulin-dependent protein kinase IV (CaMKIV) are two proteins encoded by the Camk4 gene. CaMKIV is found in multiple tissues, including brain, thymus, and testis, while calspermin is restricted to the testis. In the mouse testis, both proteins are expressed within elongating spermatids. We have recently shown that deletion of CaMKIV has no effect on calspermin expression but does impair spermiogenesis by disrupting the exchange of sperm basic nuclear proteins. The function of calspermin within the testis is unclear, although it has been speculated to play a role in binding and sequestering calmodulin during the development of the germ cell. To investigate the contribution of calspermin to spermatogenesis, we have used Cre/lox technology to specifically delete calspermin, while leaving kinase expression intact. We unexpectedly found that calspermin is not required for male fertility. We further demonstrate that CaMKIV expression and localization are unaffected by the absence of calspermin and that calspermin does not colocalize to the nuclear matrix with CaMKIV.
View details for Web of Science ID 000170349900034
View details for PubMedID 11486043
Female fertility is reduced in mice lacking Ca2+ calmodulin-dependent protein kinase IV
2000; 141 (12): 4777-4783
Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine protein kinase with limited tissue distribution. CaMKIV is highly expressed in the testis, where it is found in transcriptionally inactive elongating spermatids. We have recently generated mice deficient in CaMKIV. In the absence of CaMKIV, the exchange of sperm nuclear basic proteins in male spermatids is impaired, resulting in male infertility secondary to defective spermiogenesis. The involvement of CaMKIV in female fertility has not been addressed. Here we report that female fertility is markedly reduced in CaMKIV-deficient mice due to impaired follicular development and ovulation. CaMKIV is expressed in the ovary, where it is localized in granulosa cells. We further find that in cultured granulosa cells, CaMKIV expression and subcellular localization are hormonally regulated. As granulosa cells differentiate, CaMKIV levels decrease and the kinase translocates from the nucleus into the cytoplasm. Our results demonstrate a critical role for CaMKIV in female reproduction and point to a potential function in granulosa cell differentiation.
View details for Web of Science ID 000165360900056
View details for PubMedID 11108293
Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4
2000; 25 (4): 448-452
Ca2+/calmodulin-dependent protein kinase IV (Camk4; also known as CaMKIV), a multifunctional serine/threonine protein kinase with limited tissue distribution, has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. In the mouse testis, however, Camk4 is expressed in spermatids and associated with chromatin and nuclear matrix. Elongating spermatids are not transcriptionally active, raising the possibility that Camk4 has a novel function in male germ cells. To investigate the role of Camk4 in spermatogenesis, we have generated mice with a targeted deletion of the gene Camk4. Male Camk4-/- mice are infertile with impairment of spermiogenesis in late elongating spermatids. The sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine-2 and prolonged retention of transition protein-2 (Tnp2) in step-15 spermatids. Protamine-2 is phosphorylated by Camk4 in vitro, implicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in mammalian male germ cells. Defects in protamine-2 have been identified in sperm of infertile men, suggesting that our results may have clinical implications for the understanding of human male infertility.
View details for Web of Science ID 000088615000024
View details for PubMedID 10932193
Ca2+/calmodulin-dependent protein kinase IV is expressed in spermatids and targeted to chromatin and the nuclear matrix
JOURNAL OF BIOLOGICAL CHEMISTRY
2000; 275 (11): 7994-7999
Ca(2+)/calmodulin-dependent protein kinase IV and calspermin are two proteins encoded by the Camk4 gene. Both are highly expressed in the testis, where in situ hybridization studies in rat testes have demonstrated that CaMKIV mRNA is localized to pachytene spermatocytes, while calspermin mRNA is restricted to spermatids. We have examined the expression patterns of both CaMKIV and calspermin in mouse testis and unexpectedly find that CaMKIV is expressed in spermatogonia and spermatids but excluded from spermatocytes, while calspermin is found only in spermatids. CaMKIV and calspermin expression in the testis are stage-dependent and appear to be coordinately regulated. In germ cells, we find that CaMKIV is associated with the chromatin. We further demonstrate that a fraction of CaMKIV in spermatids is hyperphosphorylated and specifically localized to the nuclear matrix. These novel findings may implicate CaMKIV in chromatin remodeling during nuclear condensation of spermatids.
View details for Web of Science ID 000085913300078
View details for PubMedID 10713118
- Pharmacology Crashing the boards: a user friendly study guide for the USMLE step 1 Lippincott Williams and Wilkins. 1999; 2: 31–47