Regulation of Fibulin-2 Gene Expression by Integrin α3β1 Contributes to the Invasive Phenotype of Transformed Keratinocytes

ABSTRACT An important step in Salmonella enterica serovar Typhimurium virulence is the ability to invade the intestinal epithelium. The invasion process requires a large number of genes encoded on Salmonella pathogenicity island 1 (SPI-1) at centisome 63 as well as genes located in other positions throughout the chromosome. Expression of the invasive phenotype is tightly regulated by environmental cues that are processed by a complex regulatory scheme. A central player in the invasion regulatory pathway is the HilA protein, which is transcriptional activator belonging to the OmpR/ToxR family. A number of positive regulators (hilC, hilD, fis, sirA/barA, csrAB, phoBR, fadD, envZ/ompR, and fliZ) and negative regulators (hha, hilE, lon, ams, phoP c and pag) have been identified that are able to alter expression of hilA transcription. Recent work has found that hilA transcription requires the HilD protein for activation. Other work has emphasized the importance of HilE as a negative regulator of hilA. Overexpression of hilE superrepresses hilA transcription, as well as the invasive phenotype. Two-hybrid experiments suggest that HilE exerts its regulatory influence on hilA through protein-protein interactions with HilD as the protein does not bind to the hilA promoter nor does it affect hilD transcription. As it seems likely that hilE plays an important role in translating environmental signals into invasion gene regulation, we have attempted to identify how the hilE gene itself is regulated. Our results indicate that the fimYZ genes, response regulatory proteins involved in type 1 fimbrial gene expression and recently implicated in motility gene regulation, are important activators of hilE expression. These findings indicate that invasion gene expression is coregulated with motility and adherence and provide experimental evidence that the expression of these virulence phenotypes is a subset of the overall regulation of bacterial physiology.

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Comparative use of CRISPR and RNAi to modulate integrin α3β1 in triple negative breast cancer cells reveals that some pro-invasive/pro-metastatic α3β1 functions are independent of global regulation of the transcriptome

PLoS ONE ◽

10.1371/journal.pone.0254714 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0254714

Author(s):

James Kenney ◽

Abibatou Ndoye ◽

John M. Lamar ◽

C. Michael DiPersio

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Tumor Growth ◽

Cancer Cells ◽

Triple Negative Breast Cancer ◽

Cell Invasion ◽

Triple Negative ◽

Rnai Technology ◽

Integrin Α3β1

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.

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Gene expression of the invasive phenotype of TNF-α-treated MCF-7 cells

Biomedicine & Pharmacotherapy ◽

10.1016/j.biopha.2009.04.032 ◽

2009 ◽

Vol 63 (6) ◽

pp. 421-428 ◽

Cited By ~ 31

Author(s):

Yongmei Yin ◽

Xiaofeng Chen ◽

Yongqian Shu

Keyword(s):

Gene Expression ◽

Invasive Phenotype ◽

Tnf Α ◽

Mcf 7

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HilE Interacts with HilD and Negatively Regulates hilA Transcription and Expression of the Salmonella enterica Serovar Typhimurium Invasive Phenotype

Infection and Immunity ◽

10.1128/iai.71.3.1295-1305.2003 ◽

2003 ◽

Vol 71 (3) ◽

pp. 1295-1305 ◽

Cited By ~ 70

Author(s):

M. Aaron Baxter ◽

Thomas F. Fahlen ◽

Rebecca L. Wilson ◽

Bradley D. Jones

Keyword(s):

Gene Expression ◽

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Gastrointestinal Disease ◽

Pathogenicity Island ◽

Specific Gene ◽

Negative Effects ◽

Invasive Phenotype ◽

Serovar Typhimurium ◽

Two Hybrid

ABSTRACT The ability of Salmonella enterica serovar Typhimurium to traverse the intestinal mucosa of a host is an important step in its ability to initiate gastrointestinal disease. The majority of the genes required for this invasive characteristic are encoded on Salmonella pathogenicity island 1 (SPI1), and their expression is controlled by the transcriptional activator HilA, a member of the OmpR/ToxR family of proteins. A variety of genes (hilC, hilD, fis, sirA/barA, csrAB, phoB, fadD, envZ/ompR, fliZ, hilE, ams, lon, pag, and hha) have been identified that exert positive or negative effects on hilA expression, although the mechanisms by which these gene products function remain relatively unclear. Recent work indicates that the small DNA-binding protein, Hha, has a significant role in repressing hilA transcription and the invasive phenotype, particularly in response to osmolarity signals. We have characterized the Salmonella-specific gene, hilE, and found that it plays an important regulatory role in hilA transcription and invasion gene expression. Mutation of hilE causes derepression of hilA transcription, and overexpression of hilE superrepresses hilA expression and the invasive phenotype. Bacterial two-hybrid experiments indicate that the HilE protein interacts with HilD, suggesting a possible mechanism for HilE negative regulation of hilA gene expression and the Salmonella invasive phenotype. Finally, we have found that the hilE gene resides on a region of the serovar Typhimurium chromosome that has many characteristics of a pathogenicity island.

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