Integrin signaling potentiates transforming growth factor-beta 1 (TGF-ß1) dependent down-regulation of E-Cadherin expression - Important implications for epithelial to mesenchymal transition (EMT) in renal cell carcinoma.

Signal transduction through the transforming growth factor-beta 1 (TGF-ß1) pathway affects epithelial to mesenchymal transition (EMT), partly by modulation of E-Cadherin expression. The concurrent impact of extracellular matrix driven regulation of integrin signaling on EMT has not been well characterized. We assessed the cumulative effect and molecular mechanisms of TGF-ß1 and integrin signal transduction on E-Cadherin in a renal cell cancer (RCC) model. Stimulation of RCC cells with TGF-ß1 demonstrated a three-fold increased expression of integrin αv. A ligand of integrin αv-ß3, (cyclopentapeptide containing Arginyl-Glycyl-Aspartic acid motif, RGD), was used to mimic integrin signaling. Treatment of cells with RGD and TGF-ß1 demonstrated significantly greater E-cadherin depletion than either ligand alone. This cooperative action on E-Cadherin expression is regulated by transcription factor Snai1 and is followed on a cellular level by increased cellular mobility as evidenced in a wound healing assay. Subsequent silencing of potential downstream mediators of the cumulative action of RGD and TGF-ß1 was carried out by small interfering RNA transfection and confirmed by Western blotting and/or RT-PCR. SiRNA mediated silencing of FAK and PINCH1 independently abrogated the cumulative effect of RGD and TGF-ß1 on E-Cadherin expression. We have identified a novel mechanism through which extracellular matrix event transduction by integrins further augments TGF-ß1 related effects on EMT. Molecular machinery involved in the integrin αv-TGF-ß1 interplay may represent a therapeutic target in RCC.

Properties of free and occupied androgen receptor in rat skeletal muscle cytosol: effect of testosterone.

Our aim was to investigate the effect of a single testosterone (T) injection on the androgen receptor (AR) in rat skeletal muscle (SM) cytosol. The properties of AR were studied in order to establish the protocol for differential determination of free and hormone-occupied AR in SM cytosols from non-hormone-deficient animals. Using the developed ligand-exchange protocol, we demonstrated that injection of T (1 mg/kg) caused alternating changes of the total AR binding. The binding minimum (23% of the control) was measured 1 h after the injection. It was followed by pronounced and lasting elevation of the AR binding. In the control cytosols, AR complexes constituted approximately 25% of the total receptor content. Changes of their relative content immediately after T administration were consistent with rapid nuclear translocation of the AR. Inhibition of protein synthesis by cycloheximide (CHI) injection demonstrated that delayed and lasting increase of the AR binding after T injection partially depended on the stimulated protein synthesis. Altogether, the obtained evidence supports the assumption that the AR mediates elevation of its own gene expression in SM upon administration of T.

Influence of physical exercise on polyamine synthesis in the rat skeletal muscle.

BACKGROUND: Physical exercise and testosterone administration result in a series of adaptive anabolic phenomena in the skeletal muscle. The role of polyamines in these processes has been poorly explored. DESIGN: We measured the activities of polyamine-synthesising enzymes, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) and polyamine content in skeletal muscle of male rats exposed to endurance or resistance exercise, or a single testosterone treatment. Soleus muscle (consisting mainly of slow-twitching oxidative fibres-STO) and extensor digitorum longus (mainly fast-twitching glycolytic muscle fibres-FTG) were analysed for polyamine content by HPLC, and ODC and SAMDC activity. RESULTS: Both endurance and resistance exercise induced a threefold increase in endogenous testosterone production. Two hours after exercise, ODC was increased in STO fibres, returning to baseline after 24 h; in FTG fibres the increase was less prominent. An increase in SAMDC activity occurred in a more sustained manner, with its peak 8 h after exercise. Polyamines were subsequently accumulated in both skeletal muscle fibres, with a rise in putrescine concentration after 2 h, and a fall corresponding to conversion of putrescine to spermidine and spermine by SAMDC. Single dose of 17alpha-methyltestosterone resulted in a similar increase in polyamine-synthesising enzyme activities and polyamine concentrations in the skeletal muscle. CONCLUSION: Polyamine accumulation in the skeletal muscle after physical exercise is likely to occur secondary to testosterone production. Polyamines are apparently involved in the oxidative, but not in glycolytic processes related to muscle adaptation to exercise.

The effect of retabolil and training on activity of RNA polymerase in skeletal muscles.

One hundred and twenty male albino rats weighing 180--200 g were used to determine the effect of anabolic steroid hormones on adaptive changes in the synthesis of ribosomal RNA both in sedentary animals and in animals involved in a training program. One injection of Retabolil (0.1 mg/100 g body weight) increased the alpha-amanitin insensitive RNA polymerase activity of nuclei from skeletal muscles. Fourteen h after this hormone injection the enzyme activity was 45% higher than in control animals and it remained at this level for 4 days. Under these conditions a selective binding of 19-nortestosterone with cytoplasmic proteins of skeletal muscle was found. Physical training increased the RNA polymerase activity by 50% (P less than 0.05). It was found that the testosterone binding capacity of a cytoplasmic extract from trained animals was 70% greater than that of the control animals (P less than 0.05). Four injections of Retabolil during training resulted in an additional increase of RNA polymerase activity of 40% (P less than 0.05) but reduced the testosterone binding capacity of the cytoplasmic proteins that occurred with training by 21%. The findings demonstrate the effect of anabolic hormones in the regulation of RNA synthesis in skeletal muscle nuclei in the process of their adaptation to systematic physical training.