ALK1 Opposes ALK5/Smad3 Signaling and Expression of Extracellular Matrix Components in Human Chondrocytes

Abstract

ABSTRACT:Introduction: TGF-␤ is a multifunctional regulator of chondrocyte proliferation, differentiation, and extracellular matrix production. Dysregulation of TGF-␤ action has been implicated in cartilage diseases such as osteoarthritis. TGF-␤ signaling is transduced through a pair of transmembrane serine/threonine kinases, known as the type I (ALK5) and type II receptors. However, recent studies on endothelial cells have identified ALK1 as a second type I TGF-␤ receptor and have shown that ALK1 and ALK5 have opposing functions in these cells. Here we examined ALK1 expression and its regulation of TGF-␤ signaling and responses in human chondrocytes. Materials and Methods: ALK1 expression in human chondrocytes was examined by RT-PCR and Western blot. The ability of ALK1 to form complexes with other TGF-␤ receptors was determined by affinity labeling/ immunoprecipitation and by immunoprecipitation followed by Western blot. The effect of ALK1 on TGF-␤1-induced signaling and responses was determined by varying ALK1 expression levels and measuring transcriptional activity using promoter/luciferase assays, Smad1/5 and Smad3 phosphorylation, and expression of type II collagen, PAI-1, and fibronectin. Results: Our results indicate that ALK1 is expressed in human chondrocytes and that it is a component of the TGF-␤ receptor system, associating with ALK5, type II TGF-␤ receptor, endoglin, and betaglycan. Furthermore, we show that both ALK1 and ALK5 are needed for TGF-␤-induced phosphorylation of intracellular mediators Smad1/5, whereas only ALK5 is essential for TGF-␤1-induced phosphorylation of Smad3. In addition, our results show that ALK1 inhibits, whereas ALK5 potentiates, TGF-␤-induced Smad3-driven transcriptional activity and the expression of PAI-1, fibronectin, and type II collagen in chondrocytes. Conclusions: Our results suggest that ALK1 and ALK5 display opposing functions in human chondrocytes, implicating an essential role for ALK1 in the regulation of TGF-␤ signaling and function in these cells.

The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity

“…Ingenuity analysis predicted that the two most significant upstream regulators of the differential genes were TGF-β (inhibited) and TP53 (inhibited). TGF-β signaling plays critical roles in maintaining cartilage metabolic homeostasis and structural integrity (29)(30)(31)(32)(33). TGF-β functions via both the ALK5/SMAD-2/3 and the ALK1/SMAD-1/5/8 signaling pathways, which often ures 7 and 8; and Table 1).…”

“…play antagonizing roles (31,32). Interestingly, in aged and OA cartilage, TGF-β signaling is known to be skewed toward the ALK1/SMAD-1/5/8 pathway, contributing to the catabolic responses of aged and arthritic chondrocytes to TGF-β (29,31,32).…”

“…Interestingly, in aged and OA cartilage, TGF-β signaling is known to be skewed toward the ALK1/SMAD-1/5/8 pathway, contributing to the catabolic responses of aged and arthritic chondrocytes to TGF-β (29,31,32). Indeed, RNA-seq showed that Alk1 (Acvrl1) levels were rhythmic in WT mice and constantly upregulated in cKO mice at the night time points (Supplemental Figure 12), while Alk5 (Tgfbr1) levels remained unchanged (not shown), suggesting an increase of the Alk1/Alk5 ratio in cKO cartilage.…”

Transforming Growth Factor β1 (TGF-β1) Activates Hepcidin mRNA Expression in Hepatocytes

“…Receptors-TGF-␤1-triggered Smad1/5/8 phosphorylation is mediated by ALK1/ALK5 complexes in chondrocytes and endothelial cell (43,50,51), and heterodimers of ALK5 and ALK2/ALK3 were identified in epithelial cells, epithelium-derived tumor cells, and fibroblasts (29). However, some results contradicting the involvement of BMP receptors were reported (27,46).…”

Beyond VEGF: The NOTCH and ALK1 Signaling Pathways as Tumor Angiogenesis Targets

“…6B). Several subsequent studies have shown that immortalized or transformed cells could display a non-canonical "cross-over" TGF signaling pathway leading to SMAD1-5 phosphorylation via BMP Type I receptors (Daly et al, 2008;Finnson et al, 2008;Liu et al, 2009). This transition to "crossover" TGF/SMAD1-5 signaling from "canonical" TGF/SMAD2-3 signaling could explain how cancer cells switch from an anti-proliferative to a pro-invasive TGF response in the course of malignant cellular transformation (Liu et al, 2009).…”