Rankl genetic deficiency and functional blockade undermine skeletal stem and progenitor cell differentiation.

BACKGROUND: Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. METHODS: To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl-/-) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). RESULTS: We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl-/- mice as compared to wild-type. Rankl-/- mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl-/- SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. CONCLUSIONS: We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance.

The direct impact of pegvisomant on osteoblast functions and bone development.

PURPOSE: Acromegaly is a chronic disease characterized by growth hormone (GH) hypersecretion, usually caused by a pituitary adenoma, resulting in elevated circulating levels of insulin-like growth factor type I (IGF-I). Pegvisomant (PEG), the GH-receptor (GHR) antagonist, is used in treating acromegaly to normalize IGF-I hypersecretion. Exposure to increased levels of GH and IGF-I can cause profound alterations in bone structure that are not completely reverted by treatment of GH hypersecretion. Indeed, there is evidence that drugs used for the treatment of acromegaly might induce direct effects on skeletal health regardless of biochemical control of acromegaly. METHODS: We investigated, for the first time, the effect of PEG on cell proliferation, differentiation, and mineralization in the osteoblast cell lines MC3T3-E1 and hFOB 1.19 and its potential impact on bone development in zebrafish larvae. RESULTS: We observed that PEG did not affect osteoblast proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization. After PEG treatment, the analysis of genes related to osteoblast differentiation showed no difference in Alp, Runx2, or Opg mRNA levels in MC3T3-E1 cells. GH significantly decreased cell apoptosis (- 30 ± 11%, p < 0.001) and increased STAT3 phosphorylation; these effects were suppressed by the addition of PEG in MC3T3-E1 cells. GH and PEG did not affect Igf-I, Igfbp2, and Igfbp4 mRNA levels in MC3T3-E1 cells. Finally, PEG did not affect bone development in zebrafish larvae at 5 days post-fertilization. CONCLUSION: This study provides a first evidence of the impact of PEG on osteoblast functions both in vitro and in vivo. These findings may have clinically relevant implications for the management of skeletal health in subjects with acromegaly.

Direct effects of octreotide on osteoblast cell proliferation and function.

PURPOSE: Octreotide (OCT) is a first-generation somatostatin analog (SSA) used in the treatment of acromegaly and neuroendocrine tumors (NETs). In both diseases, OCT interacts with somatostatin receptors 2 and 5 (SSTR2 and SSTR5), inhibiting hormone hypersecretion and cell proliferation. Skeletal health is an important clinical concern in acromegaly and NETs, since acromegalic osteopathy and NET bone metastasis occur in a remarkable number of patients. While OCT's effect on NET and pituitary cells has been extensively investigated, its direct action on bone cells remains unknown. METHODS: Here, we investigated OCT direct effects on cell proliferation, differentiation, mineralization, and chemoattractant capacity of murine primary osteoblasts and osteoblast cell line MC3T3-E1. RESULTS: OCT inhibited osteoblasts and MC3T3-E1 cell proliferation (- 30 ± 16%, and - 22 ± 4%, both p < 0.05 vs control) and increased MC3T3-E1 cell apoptosis (+ 76 ± 32%, p < 0.05 vs control). The anti-proliferative action of OCT was mediated by SSTR2 and SSTR5 in MC3T3-E1, while its pro-apoptotic effect was abrogated in SSTR2-silenced cells. The analysis of genes related to the early and late phases of osteoblast differentiation showed that OCT did not affect Alp, Runx2, Bglap, Spp1, and Sost levels in MC3T3-E1 cells. Similarly, OCT did not affect ALP activity, mineralization, and osteoclastogenic induction. Finally, Vegfa expression decreased in OCT-treated MC3T3-E1 cells and OCT inhibited pancreatic NET cell migration toward the osteoblast-conditioned medium. CONCLUSION: This study provides the first evidence of the direct action of OCT on osteoblasts which may have clinically relevant implications for the management of skeletal health in subjects with acromegaly and metastatic NETs.