Attenuation of the transforming growth factor beta-signaling pathway in chronic venous ulcers

Abstract

Transforming growth factor beta (TGFbeta) is important in inflammation, angiogenesis, reepithelialization and connective tissue regeneration during wound healing. We analyzed components of TGFbeta signaling pathway in biopsies from 10 patients with nonhealing venous ulcers (VUs). Using comparative genomics of transcriptional profiles of VUs and TGFbeta-treated keratinocytes, we found deregulation of TGFbeta target genes in VUs. Using quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis, we found suppression of TGFbeta RI, TGFbeta RII and TGFbeta RIII, and complete absence of phosphorylated Smad2 (pSmad2) in VU epidermis. In contrast, pSmad2 was induced in the cells of the migrating epithelial tongue of acute wounds. TGFbeta-inducible transcription factors (GADD45beta , ATF3 and ZFP36L1) were suppressed in VUs. Likewise, genes suppressed by TGFbeta (FABP5, CSTA and S100A8) were induced in nonhealing VUs. An inhibitor of Smad signaling, Smad7 was also downregulated in VUs. We conclude that TGFbeta signaling is functionally blocked in VUs by downregulation of TGFbeta receptors and attenuation of Smad signaling resulting in deregulation of TGFbeta target genes and consequent hyperproliferation. These data suggest that application of exogenous TGFbeta may not be a beneficial treatment for VUs.

Figure 1

Deregulation of TGFβ RI, TGFβ RII and TGFβ RIII in VUs. (A) Expression levels of TGFβ RI, TGFβ RII and TGFβ RIII by qPCR. Mean values are represented after normalization to the expression level of HPRT1. Error bars indicate mean ± SD. *Statistically significant differences between VUs and healthy skin were defined as P < 0.05 (A). Immunofluorescence microscopy of healthy skin shows TGFβ RI staining in the basal layer of epidermis. TGFβ RI is absent in VUs (B). TGFβ RII is expressed throughout the epidermis of healthy skin, but not in VUs (C). TGFβ RIII is downregulated in VUs (D).

Figure 2

Expression of Smad2, Smad3, Smad4 and Smad7 in VUs and healthy skin. Real-time qPCR results for the expression of Smad2 (A), Smad3 (B), Smad4 (C) and Smad7 (D). Error bars indicate mean ± SD. *Statistically significant differences between VUs and healthy skin were defined as P < 0.05.

Figure 3

Phosphorylated Smad2 is absent and Smad7 does not contribute to the attenuation of TGFβ signaling in VUs. (A) Immunohistochemistry shows nuclear pSmad2 in basal keratinocytes in healthy skin, in acute wounds immediately upon wounding and in the migrating epithelial tongue 48 h after wounding. In contrast, no nuclear pSmad2 was observed in keratinocytes of VUs. (B) Smad7 is predominantly expressed in the basal layer in healthy skin and at the acute wound edge immediately after wounding. It is upregulated in migrating epithelial tongue 48 h after wounding and downregulated in VUs.

Figure 4

ATF3, GADD45β and ZFP36L1 are suppressed while FABP5, S100A8 and CSTA are induced in VUs. mRNA levels of ATF3, GADD45β , ZFP36L1 (A, B) and mRNA levels of FABP5, S100A8 and CSTA (C, D) in TGFβ-treated keratinocytes and VUs measured by qPCR. Error bars indicate mean ± SD.

Figure 5

Attenuation of TGFβ signaling in VU. Top, histology of healthy skin. Cartoon summarizes a simplified signaling cascade in healthy epidermis. Bottom, histology of VU. In addition to decreased levels of TGFβ , downregulation of receptors followed by subsequent loss of pSmad2 leads to deregulation of TGFβ target genes and hyperproliferative epidermis of nonhealing VU.