Adipose tissue plays a crucial role in systemic energy homeostasis, and its dysfunction contributes to obesity and related cardiovascular diseases. Bone morphogenic protein (BMP) 8B, part of the Transforming Growth Factor-β (TGF-β) superfamily, is key in regulating energy balance via activating brown adipose tissue (BAT) thermogenesis, presenting a potential target for obesity treatment. However, the molecular mechanisms underlying BMP8B function in BAT remain unclear. It is reported that BMP8B signals through both SMAD1/5/8 and SMAD2/3 pathways in several cell types, but the impact of such unusual signalling specificity in adipocyte function is unknown. Thus, investigating BMP8B functions is crucial, both for therapeutic and molecular biology insights. My PhD project aims to elucidate the molecular mechanisms of BMP8B processing, regulation and signalling in adipocytes.

My findings indicate that BMP8B secretion by mammalian cells is limited, partly due to interactions with cell-surface and matrix-bound extracellular matrix proteins like proteoglycans. This interaction likely involves the prodomain, as substituting BMP8B's prodomain with that of BMP10 enhances secretion. ProBMP8B is mainly cleaved at a consensus pro-protein convertase site near the mature ligand domain (R₂₆₀TRR₂₆₃↓), with alternative cleavage sites used when the primary site is unavailable, suggesting possible alternative processing of BMP8B. BMP8B from three commercial sources does not elicit either SMAD1/5 or SMAD2/3 phosphorylation in adipocytes, whereas BMP8B I produced in-house activates the SMAD1/5/8 but not the SMAD2/3 pathway. This activity was confirmed not to result from contamination with other TGF-β ligands. Preliminary data indicates that BMP8B's function in brown adipocytes is mediated through the type I receptor ALK3 and can be inhibited by the extracellular BMP ligand trap, Crossveinless 2.

In my final year, my thesis expanded to compare BMP4, BMP9, and BMP10 signalling in adipocytes. BMP4 has a well-documented function in adipocytes, whereas roles for BMP9 and BMP10 are less known. Despite BMP9 and BMP10 having almost identical signalling activities in vascular endothelial cells, my results reveal distinct differences in the potencies and activities of these ligands in brown and white adipocytes. RNA sequencing (RNAseq) was performed to compare BMP4, BMP9, and BMP10 signalling in fully differentiated brown and white adipocytes, and the results show that these BMPs regulate shared BMP/SMAD and PPARγ target genes. However, their effects are distinct, influencing genes related to lipogenesis, adipogenesis, neurogenesis, and angiogenesis. Interestingly, I found that Endoglin, a known co-receptor for BMP9 and BMP10 in endothelial cells, is also highly expressed in adipocytes. Knocking out Endoglin in mouse primary brown and white preadipocytes reduced BMP9 and BMP4 signalling, uncovering a potential new aspect of BMP signalling regulation in adipocytes. In conclusion, the results presented in this thesis improve our understanding of the molecular mechanisms orchestrating BMP8B function in adipocytes, while also highlighting distinct functional roles of BMP4, BMP9, and BMP10, offering new therapeutic avenues for obesity and cardiometabolic disorders.