Cells within human skin are permanently exposed to mechanical stretching. Here we present evidence that alterations in cell shape trigger biochemical signaling via MAP kinases in human keratinocytes. In an in vitro attempt we demonstrate a fast but transient activation of extracellular signal-regulated kinases 1/2 in response to cell stretch. This activation is reversed by preincubation with functional blocking antibodies directed towards beta1-integrins. As a second member of MAP kinases, stress-activated protein kinase/c-JUN NH2-terminal kinase was activated in a slower fashion, peaking at 1 h after the initial stimulus. The delay in signal transmission suggests that extracellular signal-regulated kinases 1/2 and stress-activated protein kinase/c-JUN NH2-terminal kinase do not share the same signaling pathway. p38 was not activated by cell stretching. The contribution of cytoskeletal elements in signal perception and transduction was evaluated by selective disruption of either actin filaments, microtubules, or keratin filaments but showed no clear effect on stretch-induced activation of extracellular signal-regulated kinases 1/2 and stress-activated protein kinase/c-JUN NH2-terminal kinase. In conclusion we found evidence of a cell-shape-dependent activation of MAP kinases in human keratinocytes disclosing beta1-integrins as putative mechano-transducers. It is likely that alterations of skin mechanics in vivo underlying pathogenic processes like wound formation and healing trigger physiologic responses via the MAP kinase pathway.

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Mechanical stress is known to modulate fundamental events such as cell life and death. Mechanical stretch in particular has been identified as a positive regulator of proliferation in skin keratinocytes and other cell systems. In the present study it was investigated whether antiapoptotic signaling is also stimulated by mechanical stretch. It was demonstrated that mechanical stretch rapidly induced the phosphorylation of the proto-onco- The application of mechanical forces is a ubiquitous challenge to skin cells. Both differentiation processes and proliferation can be induced by different qualities of mechanical stimulation. In vitro studies have demonstrated that mechanical pressure gives rise to differentiation processes (1, 2), while mechanical stretch supports proliferation in human epidermal cells (3-5). These findings fit the in vivo situation where mechanical pressure provokes horny skin formation, and mechanical stretch, as present in abdominal stretching during pregnancy, induces skin enlargement. Previous in vitro studies on human skin keratinocytes have shown that mechanical stretch induces proliferation-associated signaling cascades of the mitogen-activated protein kinase pathway (4). Particularly the activation of extracellular signal-regulated kinase 1/2 may have functional aspects in stretch-mediated proliferation. As initial events in mechanotransduction, surface receptors of the integrin family are thought to recognize mechanical energy and transduce it into biological signals (6, 7). This assumption is supported by the finding that blocking of ␤ 1 integrins abrogates stretch-induced activation of extracellular signal-regulated kinase 1/2 (4).In addition to increased proliferation, it could be that induction of antiapoptotic signaling pathways may contribute to the increase in cell number in response to mechanical stretch. In particular, the antiapoptotic kinase PKB 1 /Akt is a candidate signaling molecule shown to play a key role in suppression of apoptotic cell death (8, 9). Prototypically, PKB/Akt activation is transduced by cell surface receptors in response to insulin and mitogens such as epidermal growth factor (10 -12). In addition, recent advances have shown that integrins, namely the ␤ 1 and ␤ 4 subsets, stimulate PKB/Akt phosphorylation and therefore may contribute to antiapoptosis (13,14). Proximal from surface receptors, the phosphoinositide 3-OH kinase (PI3K) conveys activation of PKB/Akt via phosphoinositide-dependent kinases. It has been demonstrated that phosphoinositide-dependent kinase-1 phosphorylates PKB/Akt at threonine 308 (15), whereas the mechanism of the serine 473 phosphorylation is still under debate (16,17). Studies regarding the impact of mechanical stimuli on PKB/Akt have been mainly carried out in endothelium-derived cells as hemodynamic changes caused by coronary circulation are known to contribute to pathophysiological processes of the blood vessel system (18 -21). In this context, vascular smooth muscle cells may also respond to mechanical stretch by activation ...

Previous results indicate that the polyphenol resveratrol inhibits cell growth of colon carcinoma cells via modulation of polyamine metabolic key enzymes. The aim of this work was to specify the underlying molecular mechanisms and to identify a possible role of transcription factor peroxisome proliferator-activated receptor ; (PPAR;). Cell growth was determined by bromodeoxyuridine incorporation and crystal violet staining. Protein levels were examined by Western blot analysis. Spermine/spermidine acetyltransferase (SSAT) activity was determined by a radiochemical assay. PPAR; ligand-dependent transcriptional activity was measured by a luciferase assay. A dominant-negative PPAR; mutant was transfected in Caco-2 cells to suppress PPAR;-mediated functions. Resveratrol inhibits cell growth of both Caco-2 and HCT-116 cells in a dose-and time-dependent manner (P < 0.001). In contrast to Caco-2-wild type cells (P < 0.05), resveratrol failed to increase SSAT activity in dominantnegative PPAR; cells. PPAR; involvement was further confirmed via ligand-dependent activation (P < 0.01) as well as by induction of cytokeratin 20 (P < 0.001) after resveratrol treatment. Coincubation with SB203580 abolished SSAT activation significantly in Caco-2 (P < 0.05) and HCT-116 (P < 0.01) cells. The involvement of p38 mitogen-activated protein kinase (MAPK) was further confirmed by a resveratrol-mediated phosphorylation of p38 protein in both cell lines. Resveratrol further increased the expression of PPAR; coactivator PGC-1A (P < 0.05) as well as SIRT1 (P < 0.01) in a dose-dependent manner after 24 hours of incubation. Based on our findings, p38 MAPK and transcription factor PPAR; can be considered as molecular targets of resveratrol in the regulation of cell proliferation and SSAT activity, respectively, in a cell culture model of colon cancer. (Cancer Res 2006; 66(14): 7348-54)

Summary Antimicrobial peptides like human β‐defensin‐2 (HBD‐2) play an important role in the innate immune system protecting the intestinal mucosa against bacterial invasion. The dietary histone deacetylase (HDAC) inhibitors sulforaphane (SFN) and butyrate have received a great deal of attention because of their ability to simultaneously modulate multiple cellular targets involved in cellular protection. In this study the influence of SFN and butyrate on HBD‐2 expression as well as the molecular pathways involved in SFN‐mediated induction of HBD‐2 were scrutinized. Treatment of Caco‐2, HT‐29 and SW480 cells with SFN led to a time‐ and dose‐dependent upregulation of HBD‐2 mRNA expression as determined by semi‐quantitative reverse transcription–polymerase chain reaction. Moreover, HBD‐2 protein production increased in response to SFN, measured by enzyme‐linked immunosorbent assay. Induction of HBD‐2 was also observed in response to butyrate. Immunofluorescence analysis revealed that the protein was localized in the cytosol. Coincubation of SFN with a vitamin D receptor (VDR), or an extracellular‐regulated kinase 1/2 or a nuclear factor‐κB inhibitor all reduced HBD‐2 mRNA upregulation. In contrast, transfection of cells with a dominant‐negative peroxisome proliferator‐activated receptor γ (PPARγ) mutant vector to inhibit PPARγ wild‐type action and inhibition of p38 mitogen‐activated protein kinase (MAPK) signalling did not affect SFN‐mediated upregulation of HBD‐2 mRNA. Moreover, SFN induced the expression of VDR, PPARγ and phosphorylated ERK1/2 but did not affect p38 MAPK activation. The data clearly demonstrate for the first time that the dietary HDAC inhibitor SFN is able to induce antimicrobial peptides in colonocytes. In this process HBD‐2 expression is regulated via VDR, mitogen‐activated protein kinase kinase/extracellular‐regulated kinase and nuclear factor‐κB signalling.