Integrin ?3?1 signaling through MEK/ERK determines alternative polyadenylation of the MMP-9 mRNA transcript in immortalized mouse keratinocytes.

ABSTRACT: Integrin ?3?1 is highly expressed in both normal and tumorigenic epidermal keratinocytes where it regulates genes that control cellular function and extracellular matrix remodeling during normal and pathological tissue remodeling processes, including wound healing and development of squamous cell carcinoma (SCC). Previous studies identified a role for ?3?1 in immortalized and transformed keratinocytes in the regulation of genes that promote tumorigenesis, invasion, and pro-angiogenic crosstalk to endothelial cells. One such gene, matrix metalloproteinase-9 (MMP-9), is induced by ?3?1 through a post-transcriptional mechanism of enhanced mRNA stability. In the current study, we sought to investigate the mechanism through which ?3?1 controls MMP-9 mRNA stability. First, we utilized a luciferase reporter assay to show that AU-rich elements (AREs) residing within the 3'-untranslated region (3'-UTR) of the MMP-9 mRNA renders the transcript unstable in a manner that is independent of ?3?1. Next, we cloned a truncated variant of the MMP-9 mRNA which is generated through usage of an alternative, upstream polyadenylation signal and lacks the 3'-UTR region containing the destabilizing AREs. Using an RNase protection assay to distinguish "long" (full-length 3'-UTR) and "short" (truncated 3'-UTR) MMP-9 mRNA variants, we demonstrated that the shorter, more stable mRNA that lacks 3'-UTR AREs was preferentially generated in ?3?1-expressing keratinocytes compared with ?3?1-deficient (i.e., ?3-null) keratinocytes. Moreover, we determined that ?3?1-dependent alternative polyadenylation was acquired by immortalized keratinocytes, as primary neonatal keratinocytes did not display ?3?1-dependent differences in the long and short transcripts. Finally, pharmacological inhibition of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway in ?3?1-expressing keratinocytes caused a shift towards long variant expression, while Raf-1-mediated activation of ERK in ?3-null keratinocytes dramatically enhanced short variant expression, indicating a role for ERK/MAPK signaling in ?3?1-mediated selection of the proximal polyadenylation site. These findings identify a novel mode of integrin ?3?1-mediated gene regulation through alternative polyadenylation.

SUBMITTER: Missan DS 

PROVIDER: S-EPMC4353714 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature