Faculty Bibliography

Osteoporosis can result from the loss of sex hormones and/or aging. Abaloparatide (ABL), an analog of parathyroid hormone-related protein (PTHrP 1-36), is the second osteoanabolic therapy approved by the US Food and Drug Administration after teriparatide (PTH 1-34). All three peptides bind PTH/PTHrP receptor type 1 (PTHR1), but the effects of PTHrP (1-36) or ABL in the osteoblast remain unclear. We show that, in primary calvarial osteoblasts, PTH (1-34) promotes a more robust cAMP response than PTHrP (1-36) and ABL and causes a greater activation of protein kinase A (PKA) and cAMP response element-binding protein (CREB). All three peptides similarly inhibited sclerostin (SOST). Interestingly, the three peptides differentially modulated two other PKA target genes, c-Fos and receptor activator of nuclear factor kappa-B ligand (RANKL), and the latter both in vitro and in vivo. Knockdown of salt-inducible kinases (SIKs) 2 and 3 and CREB-regulated transcription coactivator 3 (CRTC3), indicated that all three are part of the pathway that regulates osteoblastic RANKL expression. We also show that the peptides differentially regulate the nuclear localization of CRTC2 and CRTC3, and that this correlates with PKA activation. Moreover, inhibition of protein phosphatases 1 and 2A (PP1/PP2A) activity revealed that they play a major role in both PTH-induced RANKL expression and the effects of PTH (1-34) on CRTC3 localization. In summary, in the osteoblast, the effects of PTH (1-34), PTHrP (1-36), and ABL on RANKL are mediated by differential stimulation of cAMP/PKA signaling and by their downstream effects on SIK2 and 3, PP1/PP2A, and CRTC3.

Parathyroid hormone (PTH) acts as a regulator of calcium homeostasis and bone remodeling. Runx2, an essential transcription factor in bone, is required for osteoblast differentiation. Noncoding RNAs such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play crucial roles in regulating gene expression in osteoblasts. In this study, we investigated the effects of PTH on osteoblast differentiation via Runx2, lncRNA, and miRNA expression in human bone marrow stromal cells (hBMSCs) and human osteoblastic cells (MG63). PTH-treatment of hBMSCs for 24 h, 7 days, and 14 days stimulated Runx2 mRNA expression. Using bioinformatics tools, we identified 17 lncRNAs originating from human Runx2 gene. Among these, lnc-SUPT3H-1:16 (RUNX2-AS1:32) expression was highly up-regulated by the 7 d PTH-treatment in hBMSCs. We also identified miR-6797-5p as the putative target of lnc-SUPT3H-1:16 and Runx2 using bioinformatics tools. PTH-treatment increased the expression of miR-6797-5p in hBMSCs, and overexpression of miR-6797-5p decreased osteoblast differentiation in MG63 cells, suggesting a role for lnc-SUPT3H-1:16 as sponge molecule. A luciferase gene reporter assay identified direct targeting of miR-6797-5p with lnc-SUPT3H-1:16 and 3'UTR Runx2 in MG63 cells. Thus, PTH stimulated the expression of lnc-SUPT3H-1:16, miR-6797-5p and Runx2, and due to the sponging mechanism of lnc- SUPT3H-1:16 towards miR-6797-5p, Runx2 was protected, resulting in the promotion of osteoblast differentiation.

Histone deacetylase 4 (Hdac4) is known to control chondrocyte hypertrophy and bone formation. We have previously shown that parathyroid hormone (PTH) regulates many aspects of Hdac4 function in osteoblastic cells in vitro; however, in vivo confirmation was previously precluded by pre-weaning lethality of the Hdac4 deficient mice. To analyze the function of Hdac4 in bone in mature animals, we generated mice with osteoblast lineage-specific knockout of Hdac4 (Hdac4^ob-/-) by crossing transgenic mice expressing Cre recombinase under the control of a 2.3kb fragment of the Col1a1 promoter with mice bearing loxP-Hdac4. The Hdac4^ob-/-mice survive to adulthood and developed a mild skeletal phenotype. At 12 weeks of age, they had short, irregularly-shaped and stiff tails due to smaller tail vertebrae, with almost no growth plates. The tibial growth plate zone was also thinned and Mmp13 and Sost mRNAs were increased in the distal femurs of Hdac4^ob-/-mice. Immunohistochemistry showed that sclerostin was elevated in Hdac4^ob-/-mice, suggesting that Hdac4 inhibits its gene and protein expression. To determine the effect of PTH in these mice, hPTH (1-34) or saline were delivered for 14 days with subcutaneously implanted devices in 8-week-old female Hdac4^ob-/-and wild type (Hdac4^fl/fl) mice. Serum CTX, a marker of bone resorption, was increased in Hdac4^ob-/-mice with or without PTH treatment. Tibial cortical BV/TV, Ct.Th, and relative cortical area (RCA) were decreased in Hdac4^ob-/-mice but PTH caused no further decrease in Hdac4^ob-/-mice. Tibial trabecular BV/TV and thickness were not changed significantly in Hdac4^ob-/-mice but decreased with PTH treatment. These results indicate that Hdac4 inhibits bone resorption and has anabolic effects via inhibiting Mmp13 and Sost/sclerostin expression. Hdac4 influences cortical bone mass and thickness and knockout of Hdac4 prevents the catabolic effect of PTH in cortical bone.

Abaloparatide (ABL), a novel analog of parathyroid hormonerelated protein (PTHrP 1-36) had similar bone anabolic effects to teriparatide (PTH 1-34), but lesser bone resorption and became the second FDA-approved osteoanabolic therapy for the treatment of osteoporosis. This study aims to elucidate the effects of PTH (1-34), PTHrP (1-36), and ABL on bone remodeling in mice. Intermittent daily subcutaneous peptide injections of 80 lg/kg/day were administered to 4 month-old C57Bl/6 male mice for 6 weeks (n = 10/group). Dual Energy X-ray Absorptiometry was performed during the course of the treatment. Eighteen hours after the final injection, right femurs were harvested for lCT analyses, sera were assayed for P1NP and CTX, and tibiae were separated into cortical, trabecular, and bone marrow fractions for RT-qPCR analyses. We show that ABL resulted in a similar increase in whole body, femoral, and tibial bone mineral density (BMD) compared with PTH (1-34). lCT analyses revealed similar increases in cortical thickness with ABL and PTH (1-34). ABL was superior to PTH (1-34) and PTHrP (1-36) in stimulating greater levels of P1NP, and PTH (1-34) and ABL led to increases in CTX. Surprisingly, PTHrP (1-36) seemed to have no effect on P1NP and CTX levels, BMD or cortical thickness. RT-qPCR analyses of trabecular bone populations showed that PTH (1-34) and ABL led to a similar rise in osteoblastic gene expression. Surprisingly, PTH (1-34) and PTHrP (1-36) led to increases in osteoblastic mRNAs in bone marrow, while ABL did not. These data reveal that ABL is equivalent to PTH (1-34) in increasing BMD and bone osteoblastic gene expression. The relative serum P1NP and CTX levels showed that ABL is better able to stimulate bone formation. Taken together, this study provides greater insight into the effects of PTH (1-34), PTHrP (1-36) and ABL in bone, and confirms that ABL is an effective osteoanabolic

Transforming growth factor-beta1 (TGF-beta1), a highly abundant growth factor in skeletal tissues, stimulates matrix metalloproteinase-13 (MMP-13) expression in osteoblastic cells. MMP-13 plays a critical role in bone remodeling. Runx2, a bone transcription factor, is required for TGF-beta1-mediated stimulation of MMP-13 expression in osteoblastic cells. In this study, the molecular mechanism responsible for TGF-beta1-stimulation of MMP-13 expression via Runx2 in osteoblastic cells was elucidated. TGF-beta1 stimulated the phosphorylation of Runx2 at serine amino acids, and ERK inhibition blocked this effect in rat (UMR106-01) and human (MG-63) osteoblastic cells. Pretreatment with okadaic acid, a serine-threonine phosphatase inhibitor, increased Runx2 serine phosphorylation in osteoblastic cells. When cells were pretreated with an ERK inhibitor, TGF-beta1-mediated stimulation of MMP-13 mRNA expression decreased. Nano-ESI/LC/MS analysis identified that TGF-beta1 stimulates Runx2 phosphorylation at three serine amino acids. Transient transfection of mouse mesenchymal stem cells (C3H10T1/2) with Runx2 serine mutant constructs decreased TGF-beta1-mediated Runx2 serine phosphorylation. A luciferase reporter assay identified that TGF-beta1 stimulated MMP-13 promoter activity in these cells only in the presence of the wild Runx2 construct, and not with mutant Runx2. Thus, TGF-beta1 stimulates the phosphorylation of Runx2 at three serine amino acids, and this event is required for MMP-13 expression in osteoblastic cells. Hence, this study contributes to the knowledge of events governing bone remodeling and bone-related diseases