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Two collagen receptors, integrins (alpha)1(beta)1 and (alpha)2(beta)1 can regulate distinct functions in cells. Ligation of (alpha)1(beta)1, unlike (alpha)2(beta)1, has been shown to result in recruitment of Shc and activation of the Ras/ERK pathway. To identify the downstream signaling molecules activated by (alpha)2(beta)1 integrin, we have overexpressed wild-type (alpha)2, or chimeric (alpha)2 subunit with (alpha)1 integrin cytoplasmic domain in human osteosarcoma cells (Saos-2) lacking endogenous (alpha)2(beta)1. The chimeric (alpha)2/(alpha)1 chain formed a functional heterodimer with (beta)1. In contrast to (alpha 2/(alpha)1 chimera, forced expression of (alpha)2 integrin resulted in upregulation of (alpha)1 (I) collagen gene transcription in response to three-dimensional collagen, indicating that the cytoplasmic domain of (alpha)2 integrin was required for signaling. Furthermore, signals mediated by (alpha)2(beta)1 integrin specifically activated the p38(alpha) isoform, and selective p38 inhibitors blocked upregulation of collagen gene transcription. Dominant negative mutants of Cdc42, MKK3, and MKK4 prevented (alpha)2(beta)1 integrin-mediated activation of p38(alpha). RhoA had also some inhibitory effect, whereas dominant negative Rac was not effective. Our findings show the isoform-specific activation of p38 by (alpha)2(beta)1 integrin ligation and identify Cdc42, MKK3, and MKK4 as possible downstream effectors. These observations reveal a novel signaling mechanism of (alpha)2(beta)1 integrin that is distinct from ones previously described for other integrins.

Abstract Background The expression of PDK4 is elevated by diabetes, fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. It is previously shown that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a master regulator of energy metabolism, coactivates in cell lines pyruvate dehydrogenase kinase-4 (PDK4) gene expression via the estrogen-related receptor α (ERRα). We investigated the effects of long-term high-fat diet and physical activity on the expression of PDK4, PGC-1α and ERRα and the amount and function of mitochondria in skeletal muscle. Methods Insulin resistance was induced by a high-fat (HF) diet for 19 weeks in C57BL/6 J mice, which were either sedentary or with access to running wheels. The skeletal muscle expression levels of PDK4, PGC-1α and ERRα were measured and the quality and quantity of mitochondrial function was assessed. Results The HF mice were more insulin-resistant than the low-fat (LF) -fed mice. Upregulation of PDK4 and ERRα mRNA and protein levels were seen after the HF diet, and when combined with running even more profound effects on the mRNA expression levels were observed. Chronic HF feeding and voluntary running did not have significant effects on PGC-1α mRNA or protein levels. No remarkable difference was found in the amount or function of mitochondria. Conclusions Our results support the view that insulin resistance is not mediated by the decreased qualitative or quantitative properties of mitochondria. Instead, the role of PDK4 should be contemplated as a possible contributor to high-fat diet-induced insulin resistance.

Specific PCR detection and electron microscopy of Flavobacterium columnare revealed the risk of false-negative results in molecular detection of this fish pathogen. Since freezing and thawing destroyed the cells so that DNA was for the most part undetectable by PCR and the detection of bacteria was also weakened after prolonged enrichment cultivation of samples from infected fish.