Double-stranded RNA induces cathelicidin expression in the intestinal epithelial cells through phosphatidylinositol 3-kinase-protein kinase Cζ-Sp1 pathway and ameliorates shigellosis in mice.

Cathelicidin antimicrobial peptide is a key component of the host innate immune system. It is constitutively expressed by the intestinal epithelial cells, but induced at further higher levels by different host-derived and microbial stimuli, including the ligands for Toll-like receptors (TLRs). While the underlying mechanisms of cathelicidin expression remain incompletely understood, altered expression may be associated with gastro-intestinal infections and inflammatory diseases. We demonstrate here that viral double-stranded RNA and its synthetic analog poly(I:C) are potent and tissue-specific inducers of cathelicidin mRNA and protein expression in the mouse as well as human intestinal epithelial cells. Reporter assays showed that poly(I:C) transcriptionally regulates murine cathelicidin-related antimicrobial peptide (mCRAMP) by recruiting Sp1 transcription factor to the GC-box cis-regulatory element at -71bp of the mCRAMP putative promoter. Sp1 recruitment to the endogenous mCRAMP promoter was confirmed by chromatin immunoprecipitation (ChIP) assays. Immunoblotting, qPCR, ChIP and siRNA-mediated gene knockdown studies revealed that the activation of phosphatidylinositol 3-kinase/protein kinase Cζ pathways in poly(I:C)-stimulated cells underlies Sp1 phosphorylation and recruitment to the mCRAMP promoter, leading to enhanced transcription. We further showed that intra-rectal poly(I:C) administration in mice reduces intestinal bacterial load and mucosal inflammation following Shigella flexneri 2a infection by inducing mCRAMP expression in the colonic epithelial cells. This study reports novel regulatory mechanisms of cathelicidin expression that may be targeted to treat gastro-intestinal infections.

Polo-like kinase 1 expression is suppressed by CCAAT/enhancer-binding protein α to mediate colon carcinoma cell differentiation and apoptosis.

BACKGROUND: Human polo-like kinase 1 (PLK1), a highly conserved serine/threonine kinase is a key player in several essential cell-cycle events. PLK1 is considered an oncogene and its overexpression often correlates with poor prognosis of cancers, including colorectal cancer (CRC). However, regulation of PLK1 expression in colorectal cells was never studied earlier and it is currently unknown if PLK1 regulates differentiation and apoptosis of CRC. METHODS: PLK1 expression was analyzed by real-time PCR and western blotting. Transcriptional regulation was studied by reporter assay, gene knock-down, EMSA and ChIP. RESULTS: PLK1 expression was down-regulated during butyrate-induced differentiation of HT-29 and other CRC cells. Also, PLK1 down-regulation mediated the role of butyrate in CRC differentiation and apoptosis. We report here a novel transcriptional regulation of PLK1 by butyrate. Transcription factors CCAAT/enhancer-binding protein α (C/EBPα) and Oct-1 share an overlapping binding site over the PLK1 promoter. Elevated levels of C/EBPα by butyrate treatment of CRC cells competed out the activator protein Oct-1 from binding to the PLK1 promoter and sequestered it. Binding of C/EBPα was associated with increased deacetylation near the transcription start site (TSS) of the PLK1 promoter, which abrogated transcription through reduced recruitment of RNA polymerase II. We also found a synergistic role between the synthetic PLK1-inhibitor SBE13 and butyrate on the apoptosis of CRC cells. CONCLUSION: This study offered a novel p53-independent regulation of PLK1 during CRC differentiation and apoptosis. GENERAL SIGNIFICANCE: Down-regulation of PLK1 is one of the mechanisms underlying the anti-cancer role of dietary fibre-derived butyrate in CRC.

Suppression of Spleen Tyrosine Kinase (Syk) by Histone Deacetylation Promotes, Whereas BAY61-3606, a Synthetic Syk Inhibitor Abrogates Colonocyte Apoptosis by ERK Activation.

Spleen tyrosine kinase (Syk), a non-receptor tyrosine kinase, regulates tumor progression, either negatively or positively, depending on the tissue lineage. Information about the role of Syk in colorectal cancers (CRC) is limited, and conflicting reports have been published. We studied Syk expression and its role in differentiation and apoptosis of the colonocytes. Here, we reported for the first time that expression of two transcript variants of Syk is suppressed in colonocytes during butyrate-induced differentiation, which mediates apoptosis of HT-29 cells. Despite being a known HDAC inhibitor, butyrate deacetylates histone3/4 around the transcription start site (TSS) of Syk. Histone deacetylation precludes the binding of RNA Polymerase II to the promoter and inhibits transcription. Since butyrate is a colonic metabolite derived from undigested fibers, our study offers a plausible explanation of the underlying mechanisms of the protective role of butyrate as well as the dietary fibers against CRC through the regulation of Syk. We also report that combined use of butyrate and highly specific Syk inhibitor BAY61-3606 does not enhance differentiation and apoptosis of colonocytes. Instead, BAY completely abolishes butyrate-induced differentiation and apoptosis in a Syk- and ERK1/2-dependent manner. While butyrate dephosphorylates ERK1/2 in HT-29 cells, BAY re-phosphorylates it, leading to its activation. This study describes a novel mechanism of butyrate action in CRC and explores the role of Syk in butyrate-induced differentiation and apoptosis. In addition, our study highlights those commercial small molecule inhibitors, although attractive drug candidates should be used with concern because of their frequent off-target effects. J. Cell. Biochem. 118: 191-203, 2017. © 2016 Wiley Periodicals, Inc.

Biphasic Ccl20 regulation by Toll-like receptor 9 through the activation of ERK-AP-1 and non-canonical NF-κB signaling pathways.

BACKGROUND: Chemokines play key roles in immune homeostasis and inflammatory response. Considering the role of Ccl20 and Toll-like receptor 9 (TLR9) in gut homeostasis and inflammatory bowel disease (IBD), regulation of Ccl20 by bacterial DNA, the TLR9 ligand, merits in-depth studies. METHODS: We analyzed Ccl20 expression in various epithelial cell (EC) lines by q-PCR and ELISA. In-vivo expression was investigated in isolated murine colonocytes by immunoblotting. Transcriptional regulation of Ccl20 was studied by reporter assays, gene knock-down, electrophoretic mobility shift assay and chromatin immunoprecipitation. Activation of upstream kinases was checked by immunoblotting. RESULTS: We showed low levels of Ccl20 expression in mouse colonic ECs, but marked induction by in vivo treatment with bacterial DNA. This corroborated with persistent Ccl20 induction in different EC lines. We found involvement of MAP-kinases during the early hours after stimulation, and a novel AP-1site (-252bp) regulated the expression in colonic ECs. More importantly, mutually exclusive transcriptional regulation by AP-1 (cjun/cfos) and non-canonical NF-κB (RelB/p52) downstream of MEK-ERK and NIK-IKK-α-NF-κB2 (p100) phosphorylation, respectively was responsible for persistent Ccl20 expression in the colonic cells, while canonical NF-κB isoforms played no role. CONCLUSIONS: Persistent Ccl20 induction by TLR9 in colonic ECs involves early and delayed activation of two independent signaling pathways. This is the first report of non-canonical NF-κB activation and Ccl20 expression in the colonic ECs by TLR9. GENERAL SIGNIFICANCE: Our study will help to better understand immune regulation by Ccl20 in the intestine and may be exploited for future development of novel therapeutics against IBD.

Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms.

Toll-like receptor 5 (TLR5) expression in the intestinal epithelial cells (IECs) is critical to maintain health, as underscored by multiple intestinal and extra-intestinal diseases in mice genetically engineered for IEC-specific TLR5 knockout. A gradient of expression exists in the colonic epithelial cells from the cecum to the distal colon. Intriguingly, an identical gradient for the dietary metabolite, butyrate also exists in the luminal contents. However, both being critical for intestinal homeostasis and immune response, no studies examined the role of butyrate in the regulation of TLR5 expression. We showed that butyrate transcriptionally upregulates TLR5 in the IECs and augments flagellin-induced immune responses. Both basal and butyrate-induced transcription is regulated by differential binding of Sp-family transcription factors to the GC-box sequences over the TLR5 promoter. Butyrate activates two different protein kinase C isoforms to dephosphorylate/acetylate Sp1 by serine/threonine phosphatases and phosphorylate Sp3 by ERK-MAPK, respectively. This resulted in Sp1 displacement from the promoter and binding of Sp3 to it, leading to p300 recruitment and histone acetylation, activating transcription. This is the first study addressing the mechanisms of physiological TLR5 expression in the intestine. Additionally, a novel insight is gained into Sp1/Sp3-mediated gene regulation that may apply to other genes.

Live and heat-killed probiotic Lactobacillus casei Lbs2 protects from experimental colitis through Toll-like receptor 2-dependent induction of T-regulatory response.

Inflammatory bowel disease (IBD) is a group of inflammatory disorders of the intestine caused by dysregulated T-cell mediated immune response against commensal microflora. Probiotics are reported as therapeutically effective against IBD. However, variable efficacy of the live probiotic strains, difference in survival and persistence in the gut between the strains and the lack of insight into the mechanisms of probiotic action limit optimal therapeutic efficacy. Our aims were to evaluate the lactobacillus strains isolated from the North Indian population for the generation of regulatory cells and cytokines in the intestine, to study their effects on pro-inflammatory mediators in the mouse model of inflammatory bowel disease and to explore the underlying mechanisms of their actions. Among the selected lactobacillus strains, Lactobacillus casei Lbs2 (MTCC5953) significantly suppressed lipopolysaccharide-induced pro-inflammatory cytokine (TNF-alpha, IL-6) secretion. Both live and heat-killed Lbs2 polarized Th0 cells to T-regulatory (Treg) cells in vitro, increased the frequency of FoxP3(+) Treg cells in the mesenteric lymph nodes (MLNs) and alleviated macroscopic and histopathological features of colitis in probiotic-fed mice. Moreover, the levels of IL-12, TNF-alpha and IL-17A were suppressed, while IL-10 and TGF-beta levels were augmented in the colonic tissues of Lbs2-treated mice. The induced Treg (iTreg) cells secreted IL-10 and TGF-beta and exerted suppressive effects on the proliferation of effector T-cells. Adoptive transfer of iTreg cells ameliorated the disease manifestations of murine colitis and suppressed the levels of TNF-alpha and IL-17A. Finally, Lbs2 effects were mediated by Toll-like receptor 2 (TLR2) activation on the dendritic cells. This study identified live and heat-killed Lbs2 as putative therapeutic candidates against IBD and highlighted their Toll-like receptor 2-dependent immunomodulatory and regulatory function.