The Role of Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor (BAMBI) in PTH Signaling in Bone

Abstract

Parathyroid hormone (PTH), an 84-amino acid peptide synthesized and secreted by the parathyroid glands, plays a crucial role in calcium and phosphorus metabolism, acting on bone and the kidney. Although PTH can induce both bone catabolic and anabolic effects, depending on the duration and periodicity of exposure, daily injections increase bone density in both human and animal studies. PTH acts directly on osteoblasts and osteocytes, indirectly influencing osteoclasts through interactions with osteoblasts and osteocytes. Osteocytes constitute 90%-95% of all bone cells in normal adult bone. They play an essential role in bone homeostasis by secreting various regulators that control the activity of both osteoblasts and osteoclasts during bone modeling and remodeling processes. Additionally, osteocytes release an endocrine factor, FGF23, to regulate other organs, such as the kidneys, playing a crucial role in maintaining phosphate and vitamin D homeostasis. The Baron-Gori Lab's bulk-RNA seq database from osteocyte-enriched cell populations in response to in vivo PTH treatment in mice reported an increase in several gene expressions, with Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor (Bambi) being one of the most significantly upregulated genes. Furthermore, analysis of pooled scRNA-seq public databases of bone cells revealed that BAMBI is expressed in a relatively restricted manner in osteocytes. BAMBI is a transmembrane protein pseudo-receptor that exhibits structural homology to the transforming growth factor β type I receptor (TGF-βRI) but lacks an intracellular kinase domain. It has been identified as a common transcriptional target and mediator between the Wnt and TGF-β signaling pathways, ultimately enhancing cellular growth by promoting Wnt signaling and inhibiting the TGF-β signaling pathway. Although an increase in BAMBI expression has been associated with the pathogenesis of various human pathologies, there is a limited body of literature on BAMBI's role in bone-related events, and its impact on osteocyte biology remains unexplored. Here, we demonstrated that during the differentiation of the OmGFP66 osteocyte cell line, Bambi was expressed at basal levels and continued to increase during differentiation. Importantly, Bambi expression was elevated with PTH treatment, both in a time-dependent manner—most prominently at the 1-hour time point with PTH 50 nM—and during long intermittent treatment with PTH 50 nM for 1 hour per day over 5 days. Subsequently, we knocked down Bambi expression using shRNA. Following the knockdown, we observed an upregulation of Sost and a significant increase in Tnfsf11 (RANKL) expression. Additionally, there was a decrease in non-phosphorylated β-catenin and an increase in pSMAD2, the signaling molecules of the Wnt and TGF-β pathways, respectively. Bambi knockdown (Bambi-KD) cells exhibited impaired osteocyte mineral deposition, as assessed by Alizarin Red staining, and decreased levels of the endocrine factor FGF23, as evidenced by reduced RNA and protein levels. Furthermore, the knockdown affected osteocyte morphology, with a reduction in phalloidin-stained osteocytic dendrites and decreased expression of dendritic markers Sp7 (Osterix) and Ostn (Osteocrin). Importantly, with PTH treatment, Bambi-KD cells demonstrated a significant increase in Sost expression, mirroring the inhibition of HDAC4/5 by phosphorylation, which facilitates MEF2C-driven Sost expression. Interestingly, despite reversing PTH's typical repression of Sost, Bambi-KD had no reversed effect on Tnfsf11 (RANKL) expression. Furthermore, in contrast to the usual upregulation of Fgf23 by PTH, we observed a significant downregulation, likely attributed to PTH's inability to suppress Sost in the knockdown cells. In summary, our findings reveal that BAMBI is implicated in osteocyte biology, potentially playing pivotal roles in osteocyte signaling, functions, and dendritic morphology. Notably, BAMBI may be essential for PTH, an agent routinely used to increase bone density in osteoporotic patients, to mediate the suppression of Sost and the increase in Fgf23 in osteocytes.