βig-h3 Potentiates the Profibrogenic Effect of TGFβ Signaling on Connective Tissue Progenitor Cells Through the Negative Regulation of Master Chondrogenic Genes

Abstract

Tendons and cartilage are specialized forms of connective tissues originated from common progenitor cells. Initial stages of differentiation of these tissues are characterized by the formation of cell aggregates, which share many molecular markers. Once differentiated, these cells retain considerable plasticity, and chondral metaplasia of tendon and fibrous connective tissues and eventual ossification often accompany degenerative diseases in the adult musculoskeletal system. While this fact is of great relevance for regenerative medicine and aging biology, its molecular basis remains to be elucidated. Gene expression analysis in several physiological and experimental paradigms suggests that differentiation of tendon and cartilage is regulated by a balance in the expression of chondrogenic versus tenogenic genes in the connective tissue cell precursors. Transforming growth factor b (TGFb) may function both as a profibrogenic or as a prochondrogenic factor for embryonic limb mesoderm and mesenchymal stem cell cultures, but mice that are null for TGFb 2 and 3 lack tendons. Here, we identify big-h3 as a factor downstream TGFb signaling regulated by Smad 2 and 3, which is highly expressed in the differentiating tendons and joint capsules. Furthermore, gain-and loss-of-function experiments using limb mesoderm micromass cultures show that big-h3 downregulates the expression of cartilage master genes, including Sox9, type II collagen, and Hif-1a. Positive regulation of Sox9 and type II Collagen observed in micromass cultures grown under hypoxic conditions is prevented by exogenous administration of bIG-H3, and the antichondrogenic influence of bIG-H3 is lost after Hif-1a silencing with shRNA. Collectively, our findings indicate that big-h3 promotes the fibrogenic influence of TGFb signaling, neutralizing the prochondrogenic influence of the hypoxic-inducible factor 1 activated by the hypoxic microenvironment characteristic of limb mesenchymal aggregates.

Erg cooperates with TGF-β to control mesenchymal differentiation

“…Sca1, a marker of pluripotent mesenchymal stem cells [35], was not regulated by TGF-β (Figure 1C). The results confirm previous studies indicating TGF-β promotes a fibrous phenotype in limb mesenchymal cells and extend the studies to sclerotome [13, 29, 31]. …”

“…In addition, the cells in the culture were not round and took on a spindle shape [13]. Others have recently observed a similar response to TGF-β in limb mesenchyme grown in micromass culture [29–31]. To further characterize the effects of TGF-β on sclerotome micromass cultures we treated cells with varying concentrations of TGF-β1 or BMP4 (Figure 1A).…”

Novel contribution to clubfoot pathogenesis: The possible role of extracellular matrix proteins

“…They identified TGFβip as a factor downstream to TGFβ signaling (regulated by Smad 2 and 3), which is highly expressed in joint capsules and the differentiating tendons. Their findings indicated that TGFβip promotes the fibrogenic influence of TGFβ signaling in the pro‐fibrogenic pathway …”

“…We observed specific calcified structures in the M‐side of clubfoot for the first time (Figure ). This could bring a direct connection with overexpression of asporin (which plays a role in biomineralization), TGFβip (essential for TGFβ signaling), and the intracellular pool of TGFβ (relationships to collagen calcification) in the M‐side. Moreover Seki et al declared that overexpressed CILP suppressed TFGβ signaling in lumbal disc disease .…”

Comparative transcriptional analysis of three human ligaments with distinct biomechanical properties

“…Importantly, it has been recently shown that it potentiates profibrogenic effects on connective tissue precursors under the control of TGFβ signalling (Lorda‐Diez et al. ).…”