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Regulation of fibulin-2 gene expression by integrin ?3?1 contributes to the invasive phenotype of transformed keratinocytes.

ABSTRACT: The laminin-binding integrin ?3?1 is highly expressed in epidermal keratinocytes, where it regulates both cell-autonomous and paracrine functions that promote wound healing and skin tumorigenesis. However, the roles for ?3?1 in regulating gene expression programs that control the behaviors of immortalized or transformed keratinocytes remain underexplored. In the current study, we used a microarray approach to identify genes that are regulated by ?3?1 in immortalized keratinocytes. ?3?1-Responsive genes included several genes that are involved in extracellular matrix proteolysis or remodeling, including fibulin-2 and secreted protein acidic and rich in cysteine. However, ?3?1-dependent induction of specific target genes was influenced by the genetic lesion that triggered immortalization, as ?3?1-dependent fibulin-2 expression occurred in cells immortalized by either SV40 large T antigen or p53-null mutation, whereas ?3?1-dependent expression of secreted protein acidic and rich in cysteine occurred only in the former cells. Interestingly, quantitative PCR arrays did not reveal strong patterns of ?3?1-dependent gene expression in freshly isolated primary keratinocytes, suggesting that this regulation is acquired during immortalization. p53-null keratinocytes transformed with oncogenic RasV12 retained ?3?1-dependent fibulin-2 expression, and RNAi-mediated knockdown of fibulin-2 in these cells reduced invasion, although not their tumorigenic potential. These findings demonstrate a prominent role for ?3?1 in immortalized/transformed keratinocytes in regulating fibulin-2 and other genes that promote matrix remodeling and invasion.

SUBMITTER: Missan DS

PROVIDER: S-EPMC4134363 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Regulation of fibulin-2 gene expression by integrin α3β1 contributes to the invasive phenotype of transformed keratinocytes.

Missan Dara S DS Chittur Sridar V SV DiPersio C Michael CM

The Journal of investigative dermatology 20140402 9

The laminin-binding integrin α3β1 is highly expressed in epidermal keratinocytes, where it regulates both cell-autonomous and paracrine functions that promote wound healing and skin tumorigenesis. However, the roles for α3β1 in regulating gene expression programs that control the behaviors of immortalized or transformed keratinocytes remain underexplored. In the current study, we used a microarray approach to identify genes that are regulated by α3β1 in immortalized keratinocytes. α3β1-Responsiv ...[more]

PMID: 24694902

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Project description: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.

Project description:Integrin receptors for the extracellular matrix play critical roles at all stages of carcinogenesis, including tumor growth, tumor progression and metastasis. The laminin-binding integrin α3β1 is expressed in all epithelial tissues where it has important roles in cell survival, migration, proliferation, and gene expression programs during normal and pathological tissue remodeling. α3β1 signaling and adhesion functions promote tumor growth and metastasis in a number of different types of cancer cells. Previously, we used RNA interference (RNAi) technology to suppress the expression of the ITGA3 gene (encoding the α3 subunit) in the triple-negative breast cancer cell line, MDA-MB-231, thereby generating variants of this line with reduced expression of integrin α3β1. This approach revealed that α3β1 promotes pro-tumorigenic functions such as cell invasion, lung metastasis, and gene regulation. In the current study, we used CRISPR technology to knock out the ITGA3 gene in MDA-MB-231 cells, thereby ablating expression of integrin α3β1 entirely. RNA-seq analysis revealed that while the global transcriptome was altered substantially by RNAi-mediated suppression of α3β1, it was largely unaffected following CRISPR-mediated ablation of α3β1. Moreover, restoring α3β1 to the latter cells through inducible expression of α3 cDNA failed to alter gene expression substantially, suggesting that use of CRISPR to abolish α3β1 led to a decoupling of the integrin from its ability to regulate the transcriptome. Interestingly, both cell invasion in vitro and metastatic colonization in vivo were reduced when α3β1 was abolished using CRISPR, as we observed previously using RNAi to suppress α3β1. Taken together, our results show that pro-invasive/pro-metastatic roles for α3β1 are not dependent on its ability to regulate the transcriptome. Moreover, our finding that use of RNAi versus CRISPR to target α3β1 produced distinct effects on gene expression underlines the importance of using multiple approaches to obtain a complete picture of an integrin's functions in cancer cells.

Dataset Information

Regulation of fibulin-2 gene expression by integrin ?3?1 contributes to the invasive phenotype of transformed keratinocytes.

Publications

Regulation of fibulin-2 gene expression by integrin α3β1 contributes to the invasive phenotype of transformed keratinocytes.

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