Faculty Bibliography

Klotho deficiency is a characteristic feature of chronic kidney disease in which anemia and cardiovascular complications are prevalent. Disruption of the Klotho gene in mice results in hypervitaminosis D and a syndrome resembling accelerated aging that includes osteopenia and vascular calcifications. Given that the bone microenvironment and its cellular components considerably influence hematopoiesis, in the present study, we addressed the in vivo role of klotho in blood cell formation and differentiation. Herein, we report that genetic ablation of Klotho in mice results in a significant increase in erythropoiesis and a decrease in the hematopoietic stem cell pool size in the bone marrow, leading to impaired hematopoietic stem cell homing in vivo. These data suggest that high vitamin D levels are only partially responsible for these hematopoietic changes in Klotho-/- mice. Importantly, we found similar hematopoietic abnormalities in Klotho-/- fetal liver cells, suggesting that the effects of klotho in hematopoietic stem cell development are independent of the bone microenvironment. Finally, injection of klotho protein results in hematopoietic changes opposite to the ones observed in Klotho-/- mice. These observations unveil a novel role for the antiaging hormone klotho in the regulation of prenatal and postnatal hematopoiesis and provide new insights for the development of therapeutic strategies targeting klotho to treat hematopoietic disorders associated with aging.

Osteoclasts are specialized secretory cells of the myeloid lineage important for normal skeletal homeostasis as well as pathologic conditions of bone including osteoporosis, inflammatory arthritis and cancer metastasis. Differentiation of these multinucleated giant cells from precursors is controlled by the cytokine RANKL, which through its receptor RANK initiates a signaling cascade culminating in the activation of transcriptional regulators which induce the expression of the bone degradation machinery. The transcription factor nuclear factor of activated T-cells c1 (NFATc1) is the master regulator of this process and in its absence osteoclast differentiation is aborted both in vitro and in vivo. Differential mRNA expression analysis by microarray is used to identify genes of potential physiologic relevance across nearly all biologic systems. We compared the gene expression profile of murine wild-type and NFATc1-deficient osteoclast precursors stimulated with RANKL and identified that the majority of the known genes important for osteoclastic bone resorption require NFATc1 for induction. Here, five novel RANKL-induced, NFATc1-dependent transcripts in the osteoclast are described: Nhedc2, Rhoc, Serpind1, Adcy3 and Rab38. Despite reasonable hypotheses for the importance of these molecules in the bone resorption pathway and their dramatic induction during differentiation, the analysis of mice with mutations in these genes failed to reveal a function in osteoclast biology. Compared to littermate controls, none of these mutants demonstrated a skeletal phenotype in vivo or alterations in osteoclast differentiation or function in vitro. These data highlight the need for rigorous validation studies to complement expression profiling results before functional importance can be assigned to highly regulated genes in any biologic process.

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!

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 (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.

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.

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.