DFs and HDMECs exhibited 7- to 18-fold higher levels of TRII expression than HKs (Fig. Smad2/3, not ERK1/2, in dermal cells. Similarly, expression of the constitutively activated TRI-TD kinase activated only Smad2/3 and not ERK1/2 in epidermal cells. This study provides an explanation for why TGF selectively activates ERK1/2 in certain cell types and direct evidence for TRI-independent TRII signaling to a R-Smad-independent pathway. and anti-ERK1/2 antibody (A,B) or anti-Smad2-and anti-Smad2/3 antibodies (C). ECL results were subjected scanning densitometry to measure the ratio (fold) of ERK1/2-over total ERK1/2. (D) Comparison of 3 ng/ml TGF1-stimulated and 1 ng/ml TGF3-stimulated kinetics of ERK1/2 phosphorylation. (E) Serum-starved HKs were treated without or with TGF3 (1.0 ng/ml) or TGF (200 ng/ml) and subjected to western blot analysis, as indicated. (F) The effect of PD901 on TGF3- or PDGF-BB-stimulated ERK1/2 activation. DFs were pre-treated with PD901 (10 M) for 30 minutes (and continued presence of PD901) before addition of the growth factors. We were interested whether selective activation of ERK1/2 by TGF3 in dermal but not epidermal cells also applied to Smad2/3 phosphorylation. TGF3 activation universally induced Smad2/3 phosphorylation in DFs (Fig. 1Ca), HDMECs (Fig. 1Cc) and HKs (Fig. 1Ce), which followed a similar kinetics, with maximum Smad2/3 phosphorylation CUDC-101 between 45 and 90 moments. These results indicated that this cell-type-specific effects of TGF3 on ERK1/2 phosphorylation do not apply to TGF3-induced Smad2/3 phosphorylation in the same cells. Quantitatively, TGF1 is the most abundant TGF isoform in skin wounds (Bandyopadhyay et al., 2006). By comparison, we did not detect any significant differences in ERK1/2 phosphorylation in dermal cells in response to activation by TGF1 or TGF3, although more TGF1 than TGF3 was required (Fig. 1D). We further questioned whether the failure of TGF3 to activate ERK1/2 in epidermal cells was due to an intrinsic defect in the ERK1/2 pathway. We compared activation of ERK1/2 phosphorylation by TGF3 with that of TGF (a major serum growth factor for HK growth). Although TGF3 induced a temporal decrease in the basal phosphorylation of ERK1/2 (Fig. 1Ea, lanes 2C4 vs lane 1), TGF activation (via binding to EGFR) induced a CUDC-101 two- to threefold increase in ERK1/2 phosphorylation over the basal level (Fig. 1Ea, lanes 6C9 vs lane 1). These results demonstrate CUDC-101 that there is no intrinsic defect in the ERK1/2 pathway in epidermal cells. We also investigated whether TRII directly activates ERK1/2 or functions via MEK1. We found that PD901, a specific inhibitor of MEK1, dramatically inhibited both TGF3- and PDGF-BB (platelet-derived growth factor-BB)-stimulated ERK1/2 phosphorylation (Fig. 1F). These results suggest that the activated TRII also acts via the RasCRafCMEK1 cascade to activate ERK1/2. TRII expression determines how TGF communicates with ERK1/2 To investigate the molecular basis for differential regulation of ERK1/2 by TGF3 in dermal versus epidermal cells, we focused on the expression levels of TRII and TRI/Alk5 subunits C the first TGF-interacting proteins involved in cross-membrane signaling. Although variable levels of TRI expression were found in HKs, DFs and HDMECs (Fig. 2Aa), there was no correlation between the differences in TRI levels (Fig. 2Aa lanes 1, 3 and 4) and the selective activation of ERK1/2 in dermal, but not epidermal, Mouse monoclonal antibody to Keratin 7. The protein encoded by this gene is a member of the keratin gene family. The type IIcytokeratins consist of basic or neutral proteins which are arranged in pairs of heterotypic keratinchains coexpressed during differentiation of simple and stratified epithelial tissues. This type IIcytokeratin is specifically expressed in the simple epithelia ining the cavities of the internalorgans and in the gland ducts and blood vessels. The genes encoding the type II cytokeratinsare clustered in a region of chromosome 12q12-q13. Alternative splicing may result in severaltranscript variants; however, not all variants have been fully described cells in response to TGF3. Neura-crest-originated epidermal melanocytes (MCs) were also included as a control (Fig. 2Aa lane 2). By contrast, we found a strong correlation between TRII expression levels and ERK1/2 activation. DFs and HDMECs exhibited 7- to 18-fold higher levels of TRII expression than HKs (Fig. 2Bc, lanes 3 and 4 vs lane 1). To confirm these results, we subjected sections of normal human skin to immunostaining with three anti-TRII antibodies against unique epitopes and from three impartial commercial sources. All three anti-TRII antibodies showed stronger staining of TRII in the dermis than the epidermis (Fig. 2Cb, c and d vs a). By contrast, anti-TRI antibody showed equivalent staining of both dermis and epidermis, as we have previously shown (Bandyopadhyay et al., 2006). It should be noted that, unlike epidermis that is 90% composed of HKs, the sparse staining of TRII in the dermis displays the normal distribution of DFs in the dermis, where scattered DFs are embedded in large areas of connective tissue. Therefore, a given section could only reveal a few DFs. Open in a separate windows Fig. 2. In vitro and in vivo profiles of TRIICTRI subunit in four human skin cells. (A,B) Equalized cell lysates of HKs, DFs and HDMECs and melanocytes (MCs), were subjected to western blot analyses CUDC-101 with antibodies against TRI/Alk5 (A) or TRII (B). (C) Indirect immunofluorescence staining of normal human skin with antibodies against TRII from three impartial sources, as indicated. Solid.
