Time course of gene expression signatures in mPTECs during ex vivo culture

ABSTRACT: To further examine the gene expression of isolated primary mouse proximal tubular epithelial cells (mPTECs) during ex vivo culture, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish the key regulatory transcription factor which was significantly altered. Primary culuture of mPTECs were cultured on culture dishes for 1 and 3 days. As compared to freshly isolated mPTECs, a 1801-gene consensus signature was identified that distinguished between day 1 and and day 3 samples. Within these genes, 78 transcriptal factors were dramatically altered. Expression of three genes (KLF5, KLF4, and CCND1) from this signature was quantified in the same RNA samples by RT-PCR. The proliferation of mouse proximal tubular epithelial cells in ex vivo culture depends on matrix stiffness. Combined analysis of the microarray and experimental data revealed that Krüppel-like factor 5 (Klf5) was the most upregulated transcription factor accompanied by Krüppel-like factor 4 (Klf4) downregulation when cells on stiff matrix. These changes were reversed by soft matrix via ERK inactivation. Knockdown of Klf5 or forced-expression of Klf4 inhibited stiff matrix-induced cell spreading and proliferation, suggesting that Klf5/Klf4 act as positive/negative regulators, respectively. Moreover, stiff matrix-activated ERK increased the protein level and nuclear translocation of mechanosensitive Yes-associated protein 1 (YAP1), which is reported to prevent Klf5 degradation. Finally, in vivo model of unilateral ureteral obstruction (UUO) revealed that matrix stiffness-regulated Klf5/Klf4 is related to the pathogenesis of renal fibrosis. In the dilated tubules of obstructed kidney, ERK/YAP1/Klf5/Cyclin D1 axis were upregulated and Klf4 was downregulated. Inhibition of collagen crosslinking by lysyl oxidase inhibitor alleviated UUO-induced tubular dilatation and proliferation with preserving Klf4 and suppressing the ERK/YAP1/Klf5/Cyclin D1 axis. This study unravels a novel mechanism how matrix stiffness regulates cellular proliferation and highlights the importance of matrix stiffness-modulated Klf5/Klf4 in the regulation of renal physiological functions and fibrosis progression.

ORGANISM(S): Mus musculus

PROVIDER: GSE69217 | GEO | 2015/08/01

SECONDARY ACCESSION(S): PRJNA284917

REPOSITORIES: GEO