Here we demonstrate SDF exhibit an enhanced cell surface expression of the TGF-1 accessory receptor TGFRIII (betaglycan), but not type I (ALK1 and ALK5) or type II TGF- receptors
Here we demonstrate SDF exhibit an enhanced cell surface expression of the TGF-1 accessory receptor TGFRIII (betaglycan), but not type I (ALK1 and ALK5) or type II TGF- receptors. in the expression of these genes in normal dermal fibroblasts, whereas SDF exhibited only a modest increase. We next sought to determine if higher basal expression in SDF was a result of autocrine expression of TGF. Surprisingly basal expression was not affected by a pan-neutralizing TGF antibody. To explore if altered accessory receptor expression alone could account for these changes, we determined their effects on CCN2 promoter activity. Endoglin inhibited CCN2 promoter activity in response to TGF. TGFRIII alone or in combination with endoglin was sufficient to enhance basal CCN2 promoter activity. Thus TGF accessory receptors may play a significant role in the altered expression of fibrogenic genes in SDF. test. Statistical analysis was performed using GraphPad Prism software. Results Previously we had shown SSc dermal fibroblasts (SDF) exhibit an elevated expression in the accessory receptor, endoglin (CD105). We sought to profile Solifenacin the expression of the associated TGF signalling receptors ALK1, ALK5 and TGFRIII (Betaglycan) by fluorescence-activated cell sorting analysis on these same cells. Three healthy subjects and three SSc patients were used for these analyses. SDF exhibited significantly higher cell surface expression of the accessory receptor TGFRIII (Table?1). By contrast cell surface levels of TGF type I (ALK1 and ALK5) or type II (TGFRII) receptors were not significantly elevated compared control dermal fibroblasts (NDF). Table 1 Flow cytometry analysis of cell surface expression of receptor members of the TGF family thead th rowspan=”1″ colspan=”1″ /th th rowspan=”1″ colspan=”1″ Control (AFI) /th th rowspan=”1″ colspan=”1″ SSc (AFI) /th /thead ALK110.3 (0.5)9.9 (0.2)ALK510.3 (1.4)12.5 (0.9)TGFRII7.3 (0.8)7.5 (1.6)TGFRIII79.3 (2.5)115 (2.8)aEndoglin41 (6)58 (1.4)a Open in Solifenacin a separate window Dermal fibroblasts from three healthy control subjects and three patients with diffuse cutaneous SSc were assessed for expression of ALK1, ALK5, TGFRII, TGFRIII and endoglin by FACSCalibur, and the average fluorescence intensities (AFIs) determined by subtracting the background values from the experimental values. Values given are expressed as the mean AFI SEMa, statistically significant difference in expression values ( em p /em ? ?0.05) relative to controls TGF accessory receptors have previously been implicated in modulating cellular responses of TGFs. To evaluate the impact of enhanced cell surface expression of TGFRIII and endoglin we initially assessed the effects of exogenous TGF on the expression of two fibrogenic genes, collagen type I and CCN2 in SDF. Consistent with previous studies, basal gene transcript and protein levels of collagen type I and CCN2 were significantly elevated in SDF compared to healthy controls (Fig.?1). Addition of exogenous stimulation with TGF1 induced a significant fold increase in gene transcription IKK2 and protein expression of collagen type I and CCN2 in NDF, SDF exhibited a blunted response to TGF1 with only a modest increase in these genes (Fig.?1). Open in a separate window Fig. 1 SSc dermal fibroblasts have reduced responsiveness to exogenous TGF1. Control ( em n /em ?=?3) and SSc ( em n /em ?=?3) dermal fibroblasts were assessed for the expression of COL1A2 and CCN2 in the presence or absence of TGF-1 (1?ng/ml) after 4?h by Q-PCR ( em Upper panel /em ). Protein expression of collagen type I and CCN2 was confirmed in the cell monolayers of these cells after 24?h in the presence or absence of TGF1 (1?ng/ml) by Western blot ( em Lower Panel /em ). Protein levels were Solifenacin controlled for by levels of actin expression We postulated that the apparent blunted response to exogenous responses to TGF1, and enhanced basal expression exhibited by SDF, as determined by collagen type I and CCN2 expression, may Solifenacin be due to autocrine expression of TGF1 by SDF. To assess this the effects of the pan-neutralizing TGF antibody, 1D11 (Fig.?2) on SDF collagen type.