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Cytochrome P450c17, encoded by the CYP17 gene, is a component of the 17a-hy- droxylase/17,20-lyase enzyme complex essential for production of adrenal gluco­corticoids and androgens as well as gonadal androgens. The expression of CYP17 in adrenocortical cells is stimulated by corticotropin (ACTH) via the signal trans­duction pathway involving cAMP and protein kinase A (PKA). Thus, in addition to glucocorticoids, ACTH stimulates formation of adrenal androgens, which are known to induce transforming growth factor 3 (TGF-3) secretion. TGF-3 in turn inhibits ste­roid hormone output by attenuating both basal and ACTH-dependent expression of CYP17. The present study revealed that treatment of bovine and human H295R adrenocortical cells with androgens resulted in a decrease in the basal level of CYP17 transcript and cortisol secretion, without affecting forskolin-stimulated lev­els. We also demonstrated that in H295R cells TGF-3 inhibited both basal and forskolin-stimulated accumulation of CYP17 mRNA. Determination of promoter ac­tivity, directing luciferase reporter gene expression in H295R cells transfected with deletion fragments of bovine CYP17 promoter, indicated that the -483 to -433 bp fragment of the promoter was necessary for the inhibitory action of TGF-3 on CYP17 expression. It is concluded that in bovine and human adrenocortical cells, androgens inhibit basal CYP17 expression probably at the transcriptional level and independently of the effect of TGF-3.
We aim to demonstrate the role of Alk receptors in the response of hydrogel expansion. Chondrocytes from rat knees were cultured onto plastic and hydrogel surfaces. Alk-1 and Alk-5 were overexpressed or silenced and the effects on cells during expansion were tested and confirmed using peptide inhibitors for TGFβ. Overexpression of Alk-5 and silencing of Alk-1 led to a loss of the chondrocyte phenotype, proving that they are key regulators of chondrocyte mechanosensing. An analysis of the gene expression profile during the expansion of these modified cartilage cells in plastic showed a better maintenance of the chondrocyte phenotype, at least during the first passages. These passages were also assayed in a mouse model of intramuscular chondrogenesis. Our findings indicate that these two receptors are important mediators in the response of chondrocytes to changes in the mechanical environment, making them suitable targets for modulating chondrogenesis. Inhibition of TGFβ could also be effective in improving chondrocyte activity in aged or expanded cells that overexpress Alk-1.
The recent identification of the Smad proteins has allowed the delineation of a mechanism by which TGF-β and related growth and differentiation factors convey their signals from transmembrane receptors into nucleus. Following receptor-induced activation heteromeric Smad complexes translocate into the nucleus where they act as transcription factors. Smad proteins modulate target genes through interaction with DNA and with other nuclear factors. Disruption of signalling cascade by Smad gene aberrations may cause loss of cellular responsiveness to TGF-β and thus contribute to development of cancer.
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