Faculty Bibliography

Klotho, the anti-aging hormone, is primarily expressed in kidney and is known to control mineral ion homeostasis. Genetic inactivation of klotho in mice results in a complex phenotype including significant reduction in bone mineral density and osteoblast numbers. Mineralization and bone remodeling substantially contribute to the establishment of the marrow environment and the importance of osteoblasts in hematopoiesis is well supported. The current study was designed to address the role of klotho in hematopoiesis in vivo. To test this hypothesis, we compared changes in blood cell production and differentiation in 6-week old klotho-/- and wild-type littermates. Complete automated blood count was carried out in peripheral blood. The phenotypic analysis of hematopoietic cells was assessed by flow cytometry and the colony forming potential was evaluated using methylcellulose based colony assay. Here we identified a novel function of klotho in the regulation of hematopoiesis as klotho-/- mice exhibit severe hematopoietic defects. Peripheral blood and bone marrow from klotho-/- mice display a significant increase in erythroid populations paralleled by a consistent decrease in B-lymphocytes and changes in granulocyte /monocyte populations. Interestingly, klotho-/- mice show higher levels of circulating hematopoietic stem cells (HSCs) indicating an apparent defect in either the HSCs or in the bone marrow niche. We have further observed that the bone marrow compartment of klotho-/- mice indeed harbors very low HSCs as compared to the wild type littermates. The changes in erythropoiesis and granulopoiesis are reflected in their ability to form colonies in vitro. Our observations suggest that regulation of bone and mineral metabolism by Klotho may modulate adult steady-state hematopoiesis. As klotho-/- mice exhibit hypervitaminosis D and vitamin D receptor is known to control klotho, we are currently investigating whether vitamin D plays any role in the regulation of hematopoiesis by klotho. Furthermore, !

Fibroblast growth factor-23 (FGF-23) is a bone-derived hormone which regulates phosphate homeostasis and bone mineralization. Mice deficient in Fgf-23 (Fgf-23 ) exhibit significantly elevated levels of serum phosphate and vitamin D, reduced bone mineralization and severe growth retardation. Impaired bone mineralization and changes in the bone marrow microenvironment are reported to disrupt normal hematopoiesis in mice. Our current study examines a novel role for Fgf-23 as a key regulator of hematopoiesis. Here, we analyzed 6-week-old Fgf-23 and wild-type (WT) littermate mice and characterized their hematopoietic cellular composition using automated complete blood counts and flow cytometry in peripheral blood and bone marrow. Hematology data revealed increased numbers of red blood cells and decreased numbers of white blood cells in peripheral blood of Fgf-23 mice. Moreover, flow cytometry analysis showed a reduction in B-lymphopoiesis in peripheral blood and bone marrow of Fgf-23 mice compared to WT littermates. Our recent findings furthermore suggest that Fgf-23 mice exhibit changes in T-lymphocytes, erythrocytes, and hematopoietic stem cell progenitors (HSC) in the bone marrow. In vitro colony-forming unit assays support changes in HSC function in the bone marrow of our Fgf-23 mice. In addition, we have found that exogenous administration of FGF-23 protein in WT mice results in opposite hematopoietic changes than in Fgf-23 mice. We are currently investigating the mechanisms mediating the effects of Fgf-23 on hematopoiesis. Our preliminary data on fetal liver hematopoiesis indicate that the hematopoietic changes in Fgf-23 mice are most likely due to a direct effect of Fgf-23 on HSCs rather than the result of altered bone marrow niche alone. This is the first report suggesting that Fgf-23 is directly associated with changes in hematopoiesis. Since in vivo ablation of Fgf- 23 results in hypervitaminosis D, we are currently investigating whether the effects of Fgf-23 in hematopoiesis are vitamin D!

Fibroblast growth factor 23 (FGF23) is a key regulator of mineral ion homeostasis. Genetic ablation of Fgf23 in mice leads to severe biochemical disorders including elevated serum 1,25-dihydroxyvitamin D [1,25(OH)2D], hypercalcemia, hyperphosphatemia, and marked decreased PTH levels. Because PTH stimulates 1,25(OH)2D production and increases serum calcium levels, we hypothesized that ablation of PTH from the Fgf23 knockout (Fgf23-/-) mice could suppress these affects, thus ameliorating the soft tissue and skeletal anomalies in these animals. In this study, we generated a genetic mouse model with dual ablation of the Fgf23/PTH genes. The data show that deletion of PTH does suppress the markedly higher serum 1,25(OH)2D and calcium levels observed in Fgf23-/- mice and results in much larger, heavier, and more active double-knockout mice with improved soft tissue and skeletal phenotypes. On the contrary, when we infused PTH (1-34) peptide into Fgf23-/- mice using osmotic minipumps, serum 1,25(OH)2D and calcium levels were increased even further, leading to marked reduction in trabecular bone. These results indicate that PTH is able to modulate the anomalies of Fgf23-/- mice by controlling serum 1,25(OH)2D and calcium levels.

In the growth plate, the interplay between parathyroid hormone-related peptide (PTHrP) and Indian hedgehog (Ihh) signaling tightly regulates chondrocyte proliferation and differentiation during longitudinal bone growth. We found that PTHrP increases the expression of Zfp521, a zinc finger transcriptional coregulator, in prehypertrophic chondrocytes. Mice with chondrocyte-targeted deletion of Zfp521 resembled PTHrP(-/-) and chondrocyte-specific PTHR1(-/-) mice, with decreased chondrocyte proliferation, early hypertrophic transition, and reduced growth plate thickness. Deleting Zfp521 increased expression of Runx2 and Runx2 target genes, and decreased Cyclin D1 and Bcl-2 expression while increasing Caspase-3 activation and apoptosis. Zfp521 associated with Runx2 in chondrocytes, antagonizing its activity via an HDAC4-dependent mechanism. PTHrP failed to upregulate Cyclin D1 and to antagonize Runx2, Ihh, and collagen X expression when Zfp521 was absent. Thus, Zfp521 is an important PTHrP target gene that regulates growth plate chondrocyte proliferation and differentiation.

Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPK pathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPK pathway member-encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-beta-activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2). Mechanistic analysis revealed that the TAK1-MKK3/6-p38 MAPK axis phosphorylated Runx2, promoting its association with the coactivator CREB-binding protein (CBP), which was required to regulate osteoblast genetic programs. These findings reveal an in vivo function for p38beta and establish that MAPK signaling is essential for bone formation in vivo. These results also suggest that selective p38beta agonists may represent attractive therapeutic agents to prevent bone loss associated with osteoporosis and aging.

Fibroblast growth factor-23 (FGF23) is a hormone that modulates circulating phosphate (P(i)) levels by controlling P(i) reabsorption from the kidneys. When FGF23 levels are deficient, as in tumoral calcinosis patients, hyperphosphatemia ensues. We show here in a murine model that Fgf23 ablation disrupted morphology and protein expression within the dentoalveolar complex. Ectopic matrix formation in pulp chambers, odontoblast layer disruption, narrowing of periodontal ligament space, and alteration of cementum structure were observed in histological and electron microscopy sections. Because serum P(i) levels are dramatically elevated in Fgf23(-/-), we assayed for apoptosis and expression of members from the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family, both of which are sensitive to elevated P(i) in vitro. Unlike X-linked hypophosphatemic (Hyp) and wild-type (WT) specimens, numerous apoptotic osteocytes and osteoblasts were detected in Fgf23(-/-) specimens. Further, in comparison to Hyp and WT samples, decreased bone sialoprotein and elevated dentin matrix protein-1 protein levels were observed in cementum of Fgf23(-/-) mice. Additional dentin-associated proteins, such as dentin sialoprotein and dentin phosphoprotein, exhibited altered localization in both Fgf23(-/-) and Hyp samples. Based on these results, we propose that FGF23 and (P(i)) homeostasis play a significant role in maintenance of the dentoalveolar complex.

Osteoporosis and arthritis are highly prevalent diseases and a significant cause of morbidity and mortality worldwide. These diseases result from aberrant tissue remodeling leading to weak, fracture-prone bones or painful, dysfunctional joints. The nuclear factor of activated T cells (NFAT) transcription factor family controls diverse biologic processes in vertebrates. Here, we review the scientific evidence that links NFAT-regulated gene transcription to bone and joint pathology. A particular emphasis is placed on the role of NFATs in bone resorption and formation by osteoclasts and osteoblasts, respectively. In addition, emerging data that connect NFATs with cartilage biology, angiogenesis, nociception, and neurogenic inflammation are explored. The goal of this article is to highlight the importance of tissue remodeling in musculoskeletal disease and situate NFAT-driven cellular responses within this context to inspire future research endeavors.

BACKGROUND: Cementogenesis is sensitive to altered local phosphate levels; thus, we hypothesized a cementum phenotype, likely of decreased formation, would be present in the teeth of X-linked hypophosphatemic (Hyp) mice. Mutations in the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex) cause X-linked hypophosphatemia, characterized by rickets, osteomalacia, and hypomineralized dentin formation, a phenotype recapitulated in the Hyp mouse homolog. Here, we report a developmental study of tooth root formation in Hyp mouse molars, focusing on dentin and cementum. METHODS: Light and transmission electron microscopy were used to study molar tissues from wild-type (WT) and Hyp mice. Demineralized and hematoxylin and eosin-stained tissues at developmental stages 23 to 96 days postcoital (dpc) were examined by light microscopy. Immunohistochemistry methods were used to detect bone sialoprotein (BSP) distribution in Hyp and WT mouse molar tissues, and transmission electron microscopy was used to study similar molar tissues in the non-demineralized state. RESULTS: Dentin in Hyp mice exhibited mineralization defects by 33 dpc, as expected, but this defect was partially corrected by 96 dpc. In support of our hypothesis, a cementum phenotype was detected using a combination of immunohistochemistry and transmission electron microscopy, which included thinner BSP-positive staining within the cementum, discontinuous mineralization, and a globular appearance compared to WT controls. CONCLUSION: Mutations in the phosphate-regulating Phex gene of the Hyp mouse resulted in defective cementum development.