Identification of Endoglin in Rat Hepatic Stellate Cells

Abstract

Transforming growth factor ␤ (TGF-␤) signaling is mediated by the cell surface TGF-␤ type I (ALK5), type II, and the accessory type III receptors endoglin and betaglycan. Hepatic stellate cells (HSC), the most profibrogenic cell type in the liver, express ALK5, T␤RII, and betaglycan. To monitor the expression of betaglycan in HSC, we used the commercially available antibody sc-6199 in Western blot analysis. This antibody, raised against a peptide mapping at the carboxyl terminus of the human betaglycan, is claimed to be specific for betaglycan, although it is known that the C-terminal domain is highly conserved in type III receptors. Proteins recognized in HSC by sc-6199 did not match the characteristic migration pattern of betaglycan. Moreover, the determined molecular weight (M r 160) and the observed reductant sensitivity after treatment with dithiothreitol resemble those of a closely related type III receptor, endoglin (CD105). Endoglin, a disulfide-linked homodimer, is an accessory component of the TGF-␤ receptor complex and mainly expressed on endothelial cells. The presence of endoglin in HSC of rat liver was confirmed by molecular cloning of the endoglin cDNA and immunocytochemistry. The reactivity of sc-6199 with both auxiliary TGF-␤ receptors (betaglycan and endoglin) from rats was demonstrated by Western blot and immunocytochemical analysis of cells heterologously expressing these proteins. Furthermore, Northern and Western blotting revealed that both betaglycan and endoglin genes are differentially regulated in HSC and in transdifferentiated myofibroblasts (MFB). By surface labeling and immunoprecipitation experiments, we show that endoglin is found in significant amounts exposed at the plasma membrane of HSC and MFB, which is a pivotal prerequisite for binding of and signaling in response to TGF-␤. In conclusion, we hypothesize that TGF-␤ signals in HSC and MFB are tuned by two different interconnected signaling pathways, as it was previously demonstrated for endothelial cells.The transforming growth factor family of growth factors regulates a diverse set of physiologic processes including proliferation, cellular differentiation, apoptosis, and expression of extracellular matrix genes (1-3). Signaling by the three identified mammalian TGF-␤ 1 isoforms TGF-␤1, TGF-␤2, and TGF-␤3 is initiated by binding to the high affinity cell surface receptors, the accessory TGF-␤ type III receptor (T␤RIII) (4), the type II receptor (T␤RII) (5), and type I (6) receptor (T␤RI), of which T␤RII and T␤RI possess intracellular serine/threonine kinase domains. Upon binding of TGF-␤1 or TGF-␤3 to the receptor type II, the liganded receptor dimer associates with and its constitutive active kinase transphosphorylates the type I receptor at the GS domain (7). The active type I receptor in turn phosphorylates and thereby activates the intracellular signal transducers represented by the R-Smad family members 2 and 3 (2, 7, 8).Because TGF-␤2 has a lower affinity for the type II receptor (9, 10), cells sense this ligand by eithe...

Genetic Characteristics of the Human Hepatic Stellate Cell Line LX-2

“…LX-2 cells also express fibronectin, collagen type IV, collagen type I, endoglin, TGF-β receptors type II (TβRII) and I (TβRI), vimentin, desmin, α-SMA, connective tissue growth factor (CTGF), Id2, and p21 (Figure 1), all representing known pro-fibrogenic matricellular components or mediators involved in hepatic fibrogenesis 23–25. These results partially confirm and extend previous findings demonstrating that LX-2 have attributes of primary human HSC in culture [4].…”

Id1 is a critical mediator in TGF-β-induced transdifferentiation of rat hepatic stellate cells

“…This assumption is supported by data showing endoglin exposed at the plasma membrane of activated HSCs. 37 Furthermore, preliminary data suggest that, in addition, BMP-2 signaling via ALK3 represents an active pathway in HSCs, which contributes to Smad1 activation and Id1 expression. Whether BMP-2 and TGF-␤ enhance each other in this respect or whether cross-regulatory mechanisms exist remains to be determined.…”

Elevation of endoglin (CD105) concentrations in serum of patients with liver cirrhosis and carcinoma

“…CD105 modulates TGF-b signalling by interacting with the TGF-b receptor complexes [15,16] and may be important for the regulation of epithelial-mesenchymal transition, which has a pivotal role in embryonic development, tissue repair, fibrogenesis and carcinogenesis, by binding TGF-b1 [17][18][19]. In addition, the potential of CD105 to serve as a diagnostic marker for tumour-associated angiogenesis has been assessed in several tumour types [20,21].…”