Abstract
Integrin α3β1 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 α3β1 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 α3β1 through a post-transcriptional mechanism of enhanced mRNA stability. In the current study, we sought to investigate the mechanism through which α3β1 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 α3β1. 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 α3β1-expressing keratinocytes compared with α3β1-deficient (i.e., α3-null) keratinocytes. Moreover, we determined that α3β1-dependent alternative polyadenylation was acquired by immortalized keratinocytes, as primary neonatal keratinocytes did not display α3β1-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 α3β1-expressing keratinocytes caused a shift towards long variant expression, while Raf-1-mediated activation of ERK in α3-null keratinocytes dramatically enhanced short variant expression, indicating a role for ERK/MAPK signaling in α3β1-mediated selection of the proximal polyadenylation site. These findings identify a novel mode of integrin α3β1-mediated gene regulation through alternative polyadenylation.
Highlights
The integrin α3β1 is expressed highly in epithelial cells, where it is the major receptor for laminin-332 and certain other laminin isoform present in basement membranes [1]. α3β1 functions to maintain integrity of the basement membrane during embryonic development of the epidermis, and mutations in the gene that encodes the α3 integrin subunit cause basement membrane rupture and epidermal blistering in both preclinical mouse models and human patients [2,3,4]
We previously demonstrated that absence of integrin α3β1 in immortalized mouse keratinocytes (MK cells) leads to increased turnover of the matrix metalloproteinase-9 (MMP-9) mRNA transcript, indicating an important role for this integrin in controlling MMP-9 mRNA stability [16]
The 3’-untranslated region (3’-UTR) of the full-length MMP-9 mRNA transcript contains several canonical AU-rich elements (AREs) [27, 35], and previous studies in renal mesangial cells have demonstrated that nitrous oxide and interleukin-1β can alter MMP-9 mRNA stability through regulation of these AREs [14, 15]
Summary
The integrin α3β1 is expressed highly in epithelial cells, where it is the major receptor for laminin-332 and certain other laminin isoform present in basement membranes [1]. α3β1 functions to maintain integrity of the basement membrane during embryonic development of the epidermis, and mutations in the gene that encodes the α3 integrin subunit cause basement membrane rupture and epidermal blistering in both preclinical mouse models and human patients [2,3,4]. Α3β1 functions to maintain integrity of the basement membrane during embryonic development of the epidermis, and mutations in the gene that encodes the α3 integrin subunit cause basement membrane rupture and epidermal blistering in both preclinical mouse models and human patients [2,3,4]. Some α3β1-mediated tumor cell functions may be due to its ability to induce the expression of matrix metalloproteinase-9 (MMP-9) [7,8,9]. MMP-9 is an important regulator of tumor angiogenesis and invasion with a prominent role in the development of SCC and other carcinomas [10,11,12]. Studies identified mRNA stability as an important post-transcriptional mechanism of MMP-9 gene regulation in response to cytokines, growth factors, and other stimuli [13,14,15], as well as to integrin α3β1 [16]. The mechanism whereby α3β1 controls MMP-9 mRNA stability has not been determined
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