Cold exposure and capsaicin promote 1,2-dimethylhyrazine-induced colon carcinogenesis in rats correlates with extracellular matrix remodeling.

BACKGROUND: Extracellular matrix (ECM) remodeling and stiffening, which are correlated with tumor malignancy, drives tumor development. However, the relationship between ECM remodeling and rat experimental model of 1,2-dimethylhyrazine (DMH)-induced colorectal cancer (CRC) imposed by cold and capsaicin exposure remains unclear. AIM: To explore the effects of cold exposure and capsaicin on ECM remodeling and ECM enzymes in DMH-induced CRC. METHODS: For histopathological analysis, the sections of colon tissues were stained with hematoxylin and eosin, Masson's trichrome, Picrosirius red, and Weigert's Resorcin-Fuchsin to observe the remodeling of collagen and elastin. Additionally, the protein expression level of type I collagen (COL I), type 3 collagen (COL III0, elastin, matrix metalloproteinase (MMP) 1, MMP2, MMP9, and tissue-specific matrix metalloproteinase 1 (TIMP1) was assessed by immunohistochemistry. The messenger RNA (mRNA) levels of COL I, COL III, elastin, and lysyl oxidase-like-2 (LOXL2) in the colon tissues of rats was measured by reverse-transcriptase quantitative polymerase chain reaction. RESULTS: Although no differences were observed in the proportion of adenomas, a trend towards the increase of invasive tumors was observed in the cold and capsaicin group. The cold exposure group had a metastasis rate compared with the other groups. Additionally, abnormal accumulation of both collagen and elastin was observed in the cold exposure and capsaicin group. Specifically, collagen quantitative analysis showed increased length, width, angle, and straightness compared with the DMH group. Collagen deposition and straightness were significantly increased in the cold exposure group compared with the capsaicin group. Cold exposure and capsaicin significantly increased the protein levels of COL I, elastin, and LOXL2 along with increases in their mRNA levels in the colon tissues compared with the DMH group, while COL III did not show a significant difference. Furthermore, in immunohistochemical evaluations, MMP1, MMP2, MMP9, and TIMP1 staining increased in the cold exposure and capsaicin group compared with the DMH group. CONCLUSION: These results suggest that chronic cold and capsaicin exposure further increased the deposition of collagen and elastin in the colonic tissue. Increased COL I and elastin mRNA and protein levels expression may account for the enhanced ECM remodel and stiffness variations of colon tissue. The upregulated expression of the LOXL2 and physiological imbalance between MMP/TIMP activation and deactivation could contribute to the progression of the CRC resulting from cold and capsaicin exposure.

miR-146a and Krüppel-like factor 4 form a feedback loop to participate in vascular smooth muscle cell proliferation.

MicroRNAs are phenotypic regulators of vascular smooth muscle cells (VSMCs). In this paper, we demonstrate that miR-146a targets the Krüppel-like factor 4 (KLF4) 3'-untranslated region and has an important role in promoting VSMC proliferation in vitro and vascular neointimal hyperplasia in vivo. Silencing of miR-146a in VSMCs increases KLF4 expression, whereas overexpression of miR-146a decreases KLF4 levels. Furthermore, we demonstrate that KLF4 competes with Krüppel-like factor 5 (KLF5) to bind to and regulate the miR-146a promoter, and that KLF4 and KLF5 exert opposing effects on the miR-146a promoter. Overexpression of KLF4 in VSMCs decreases miR-146a transcription levels. By using both gain-of-function and loss-of-function approaches, we found that miR-146a promotes VSMC proliferation in vitro. Transfection of antisense miR-146a oligonucleotide into balloon-injured rat carotid arteries markedly decreased neointimal hyperplasia. These findings suggest that miR-146a and KLF4 form a feedback loop to regulate each other's expression and VSMC proliferation.

Krüppel-like factor 4 promotes differentiation by transforming growth factor-beta receptor-mediated Smad and p38 MAPK signaling in vascular smooth muscle cells.

KLF4 (Krüppel-like factor 4) has been implicated in vascular smooth muscle cell (VSMC) differentiation induced by transforming growth factor beta (TGF-beta). However, the role of KLF4 and mechanism of KLF4 actions in regulating TGF-beta signaling in VSMCs remain unclear. In this study, we showed that TGF-beta1 inhibited cell cycle progression and induced differentiation in cultured rat VSMCs. This activity of TGF-beta1 was accompanied by up-regulation of KLF4, with concomitant increase in TbetaRI (TGF-beta type I receptor) expression. KLF4 was found to transduce TGF-beta1 signals via phosphorylation-mediated activation of Smad2, Smad3, and p38 MAPK. The activation of both pathways, in turn, increased the phosphorylation of KLF4, which enabled the formation of KLF4-Smad2 complex in response to TGF-beta1. Chromatin immunoprecipitation studies and oligonucleotide pull-down assays showed the direct binding of KLF4 to the KLF4-binding sites 2 and 3 of the TbetaRI promoter and the recruitment of Smad2 to the Smad-responsive region. Formation of a stable KLF4-Smad2 complex in the promoter's Smad-responsive region mediated cooperative TbetaRI promoter transcription in response to TGF-beta1. These results suggest that KLF4-dependent regulation of Smad and p38 MAPK signaling via TbetaRI requires prior phosphorylation of KLF4 through Smad and p38 MAPK pathways. This study demonstrates a novel mechanism by which TGF-beta1 regulates VSMC differentiation.