| Objectives:Programmed cell death (PDCD) 4 is a critical tumor suppressor. PDCD4 is proved to be up-regulated during apoptosis and has been identified to play an inhibitory role in the regulation of various proteins at transcriptional level and translational level. PDCD4 displays effective influence on tumor growth, invasion and metastasis. Furthermore, several researches have testified the vital function of PDCD4 in inflammatory diseases. A few of studies have confirmed that Pdcd4-deficient mice are resistant to type 1 diabetes, experimental allergic encephalitis, septic shock, and high fat-induced inflammation. Our recent researches have proved that PDCD4 inhibits inflammation for its deficiency promotes lipopolysaccharide (LPS)-and D-galactosamine-induced acute liver injury. Thus, the exact role of PDCD4 in inflammation process remains to be investigated.Inflammatory bowel diseases (IBDs) are important inflammatory diseases, and the effect of PDCD4 in IBDs is unclear. IBDs are chronic intestinal inflammation, including 2 main types, Crohn’s disease (CD) and ulcerative colitis (UC). The etiology of IBD has not been testified. The possible mechanisms include genetic factors, damage of the epithelial barrier of the gut, alteration of intestinal microbiota, and immune dysregulation. IBDs elevate the risk of colorectal carcinoma, especially the ulcerative colitis. The exact mechanism of the induction effect of colitis on colorectal carcinoma is not clear. Studies demonstrate that it may be due to the promotion effect of chronic inflammation on the neoplastic transformation of intestinal mucous.Recent researches revealed that atherosclerosis is a chronic inflammatory disease caused by metabolism disorders, and various inflammatory cytokines and immunocytes participated in the development of atherosclerosis. Furthermore, recent studies demonstrated that perivascular adipose tissue (PVAT) played a critical role during the process of atherosclerosis. PVAT was considered as a simply protective structure surrounding the vascular. However, latest researches suggest that PVAT is able to secrete series of adipokines, chemokines and hormone-like factors that exhibits metabolic activity, and thus affect the function of cardiovascular system and the development of related diseases. Our previous research demonstrated that PDCD4 promotes atherosclerosis and enhances the chronic inflammation in vascular wall during the process of atherogenesis in mice. As PVAT exhibited important influence on vascular disease, PDCD4 may also regulate vascular inflammatory diseases by affecting the PVAT and its inflammatory phenotype.In order to elucidate the role of PDCD4 in IBD and colitis-associated colorectal carcinoma, and the effect of PDCD4 on PVAT and its inflammatory phenotype, we will investigate these questions from the following two aspects:(1) The role of PDCD4 in colitis and colitis-associated colorectal carcinoma and the molecular mechanisms in mice. Pdcd4 deficiency aggravates colitis and colitis-associated colorectal cancer via promoting IL-6/STAT3 pathway in mice. (2) The role of PDCD4 on PVAT inflammatory phenotype. PDCD4 affects the phenotype of PVAT and the white adipose tissue (WAT) transformation of PVAT, and thus affects vascular diseases of adjacent blood vessels.Methods1. The role of PDCD4 in colitis and colitis-associated colorectal carcinoma and the molecular mechanisms in mice.(1) Establish acute colitis model in Pdcd4-/- mice and wild type (WT) mice. Measure body weight, fecal occult blood and calculate disease activity index (DAI). Observe pathological change and calculate inflammatory score of mice colitis tissue after H&E staining.(2) Establish colitis-associated colorectal carcinoma model in Pdcd4-/- mice and WT mice by AOM and DSS treatment. Immunohistochemichal staining was performed to detect the change of expression of PDCD4 during different stage of the course of disease. Observe pathological change and calculate inflammatory score after H&E staining, analyze the size, number and location of tumors.(3) Extract RNA from colitis tissue and synthesize cDNA, detect the expression of IL-6 and TNF-a by real-time PCR. Culture colitis tissue in vitro, and measure the levels of cytokines including IL-6 and TNF-a in supernatant by ELISA. Detect the levels of IL-6 and TNF-a in mice serum by ELISA.(4) Observe the proliferation of colonic epithelial cells during the early stage of colitis-associated colorectal carcinoma by immunohistochemichal staining of Ki-67 and BrdU.(5) Immunofluorescence staining was performed to detect the expression of p-STAT3 of different stages during the process of colitis-associated colorectal carcinoma in colon tissue.(6) Induce the production of IL-6 in peritoneal macrophages and bone marrow derived macrophages in Pdcd4-/- mice and WT mice by LPS treatment. Detect the level of IL-6 in supernatant by ELISA.(7) Transfect specific shRNA in mice colorectal carcinoma cell line to knockdown PDCD4, IL-6/STAT3 mediated cell proliferation was detected by CCK8. The effect of PDCD4 on IL-6/STAT3 pathway activation was measured by Western-blot. STAT3 inhibitor was used and repeated experiments above to confirm the role of PDCD4 in IL-6/STAT3 mediated cell proliferation.(8) Intraperitoneal inject sgp130Fc into Pdcd4-/- mice and WT mice to block IL-6/STAT3 pathway, and induce colitis-associated colorectal carcinoma with AOM and DSS treatment, observe the change of colonic epithelial cell proliferation during the early stage of colitis-associated colorectal carcinogenesis.2. The role of PDCD4 on PVAT inflammatory phenotype.(1) Establish atherosclerosis model in Apoe-*- mice and Pdcd4-/- Apoe-/-(DKO) mice after 19 weeks of high fat diet.(2) Measure body weight, observe appearance of PVAT surrounding thoracic aorta and abdominal aorta and preserve the specimens. Extract RNA from PVAT of thoracic aorta and abdominal aorta, mesenteric adipose and epididymal adipose, and synthesize cDNA. Detect the expression of various adipokines, chemokines, and cytokines by real-time PCR. Observe the morphology of PVAT surrounding thoracic aorta by H&E staining.(3) Obtain thoracic PVAT and epididymal adipose from Apoe-/- mice and DKO mice after 8 weeks of high fat diet, and transplant graft adipose tissue adjacent to left carotid artery of Apoe-/- mice after 2 weeks of high fat diet. Observe the effect of different adipose tissue on vascular diseases of adjacent artery in Apoe-/- mice.Results1. The role of PDCD4 in colitis and colitis-associated colorectal carcinoma andthe molecular mechanisms in mice.(1) Pdcd4 deficiency aggravated the DSS-induced acute colitis1) Pdcd4 deficiency exacerbated clinical symptoms of colitis in mice Pdcd4-/-mice exhibited more severe colitis and weight loss compared with WT mice. And the fecal occult blood was more severe in Pdcd4-/- mice than that in WT mice. Thus Pdcd4-/- mice had a much higher score of DAI. These results demonstrated that Pdcd4 deficiency aggravated acute colitis in mice.2) Pdcd4 deficiency aggravated the pathological change of colitis colon tissue We observed more severe pathologic change in colitis tissue of Pdcd4-/- mice than WT mice after H&E staining, including leukocyte infiltration, integrity of epithelium and edema. The inflammatory transformation occurs earlier in Pdcd4-/- mice than WT mice, and Pdcd4-/- mice had an increased inflammatory score compared with WT mice. These data demonstrated that Pdcd4 deficiency significantly exacerbated acute colitis in mice.(2) Deficiency of PDCD4 accelerated the development of colorectal carcinoma1) The expression of PDCD4 in colon decreases with the progression of colitis-associated colorectal carcinoma PDCD4 expression reduced with the progression of colitis-associated colorectal carcinoma in WT mice. PDCD4 expression was absent in tumor cells, suggesting PDCD4 is involved in colitis-associated colorectal carcinogenesis.2) The inflammation of colon during the early stage of colitis-associated colorectal carcinoma Our results revealed that both WT and Pdcd4-/- mice exhibited chronic inflammation during the early stage of colitis-associated colorectal carcinoma, and the inflammatory score was higher in Pdcd4-/- mice than that in WT mice but there was no statistically significant difference.3) The effect of PDCD4 on the size, number and location of tumors in colitis-associated colorectal carcinoma Our data demonstrated that in both WT and Pdcd4-/- mice, most tumors were located in the distal terminal of the colon. Furthermore, compared with WT mice, the size and number of tumors increased in Pdcd4-/- mice, indicating that Pdcd4 deficiency accelerates colitis-induced colorectal carcinoma.(3) Pdcd4 deficiency promoted epithelial cell proliferation in the transformative stage of carcinogenesisOur results proved that Pdcd4-/- mice exhibited an increased cell proliferation in colonic epithelium during the transformative stage of colitis-associated colorectal carcinoma.(4) Pdcd4 deficiency markedly up-regulated the expression of TNF-α and IL-6 in colon1) PDCD4 regulated TNF-a and IL-6 expression at translational level We observed that the levels of TNF-a and IL-6 in the supernatant of colonic tissue explant cultures increased significantly due to Pdcd4 deficiency. Furthermore, compared with TNF-a, the level of IL-6 was obviously higher. Furthermore, the regulation of IL-6 production by PDCD4 was more effective than the regulation of TNF-a. These results demonstrated that PDCD4 regulates the expression of TNF-a and IL-6 at translational level.2) The effect of PDCD4 on TNF-a and IL-6 expression at transcriptional level We demonstrated that PDCD4 had no effect on IL-6 expression at transcriptional level in the colon of mice, but up-regulated TNF-a expression since the 5th day of colitis at transcriptional level.3) Pdcd4 deficiency elevated the level of IL-6 in serum of mice with colitis Our observation demonstrated that Pdcd4 deficiency increased the level of IL-6 in serum from mice with colitis, confirmed that PDCD4 plays an important role in IL-6 expression.(5) Pdcd4 deficiency enhanced the activation of STAT3 in colonic epitheliumOur results demonstrated that the expression of p-STAT3 in colon was higher in Pdcd4-/- mice than that in WT mice during the process of colitis-associated colorectal carcinoma. These results proved that Pdcd4 deficiency promotes the activation of IL-6/STAT3 pathway.(6) Pdcd4 deficiency elevated IL-6 expression in macrophagesPdcd4 deficiency obviously up-regulated IL-6 expression in both peritoneal macrophages and bone marrow derived macrophages. These results suggested that the role of PDCD4 in the expression of IL-6 from immunocytes was the major mechanism of PDCD4 in the regulation of colitis and colitis-associated carcinoma.(7) Pdcd4 deficiency increased colonic epithelial cell susceptibility to IL-6/STAT3 pathway-mediated cell proliferation in vitro1) Pdcd4 deficient colonic epithelial cells exhibited enhanced proliferation Knockdown of PDCD4 by specific shRNA in mice colonic epithelial cell line had no direct effect on cell proliferation. However, after treatment of IL-6, Pdcd4 deficiency obviously enhanced colonic epithelial cells proliferation. These results proved that Pdcd4 deficiency promoted IL-6 mediated cell proliferation.2) IL-6/STAT3 pathway activation was up-regulated in Pdcd4 deficient colonic epithelial cells After IL-6 treatment, Pdcd4 deficiency enhanced the expression of p-STAT3 in colonic epithelial cell line. These data proved that Pdcd4 deficiency promoted the activation of IL-6/STAT3.3) Blockade of IL-6/STAT3 pathway in mice colonic epithelial cells eliminated the effect of PDCD4 on IL-6/STAT3 mediated cell proliferation The effect of PDCD4 on IL-6 mediated cell proliferation was eliminated after treatment of STAT3 inhibitor. These results demonstrated that PDCD4 regulates IL-6/STAT3 pathway and thus regulates IL-6/STAT3 pathway mediated cell proliferation in mice colonic epithelial cell line.(8) Blockade of IL-6/STAT3 pathway via sgp130Fc eliminated the promoting effect of Pdcd4 deficiency on colonic epithelial cell proliferation in vivoWe used sgp130Fc to block IL-6/STAT3 pathway in vivo, and the difference between WT and Pdcd4-/- mice on proliferation of colonic epithelial cells in colon at the early stage of colitis-associated colorectal carcinoma was eliminated. These data demonstrated that PDCD4 regulates colonic epithelial cell proliferation via IL-6/STAT3 pathway.2. The role of PDCD4 on PVAT inflammatory phenotype and associated inflammation.(1) PDCD4 had no effect on the weight of PVAT in Apoe-/- miceAfter 19 weeks of high fat diet, PDCD4 didn’t affect the body weight of Apoe-/-mice. Furthermore, PDCD4 had no effect on the weight and macrography of PVAT from Apoe-/- mice after 8 weeks of high fat diet.(2) Pdcd4 deficiency promoted the formation of anti-inflammatory phenotype in PVAT.1) Pdcd4 deficiency upregulated the expression of adiponectin and PPAR-y in PVAT.In Apoe-/- mice after 19 weeks of high-fat diet, Pdcd4 deficiency upregulated the expression of adiponectin in thoracic perivascular adipose tissue, and increased the expression of PPAR-y in mesenteric adipose tissue. These two adipokines are both anti-inflammatory. Pdcd4 deficiency didn’t affect the expression of leptin in Apoe-/-mice.2)PDCD4 deficiency upregulated the level of anti-inflammatory cytokine TGF-β in PVAT.Pdcd4 deficiency in Apoe-/- mice after 19 weeks of high-fat diet promoted the expression of anti-inflammatory cytokine TGF-β in mesenteric adipose tissue. And Pdcd4 deficiency didn’t affect the expression of IL-10.3)PDCD4 deficiency down-regulated the expression of proinflammatory cytokines IL-1β, IL-6, and IFN-γ in PVAT.We observed that in Apoe-/- mice after 19 weeks of high-fat diet, Pdcd4 deficiency down-regulated the expression of IL-1β in both thoracic and abdominal PVAT significantly. Furthermore, Pdcd4 deficiency reduced the level of IFN-y in abdominal PVAT, and down-regulated the expression of IL-6 in mesenteric adipose tissue. These data demonstrated that Pdcd4 deficiency inhibits the expression of proinflammatory cytokines in various depots of adipose tissue.4)Pdcd4 deficiency decreased the level of chemokine MCP-1.In Apoe-/- mice after 19 weeks of high-fat diet, Pdcd4 deficiency significantly decreased the level of chemokine MCP-1 in abdominal PVAT, and had no effect on the expression of IL-8. These results indicated that Pdcd4 deficiency in Apoe-/- mice promoted the formation of anti-inflammatory phenotype in PVAT.(3) Pdcd4 deficiency slightly attenuated the white adipose transformation in thoracic perivascular brown adipocytes.The expression of CIDEA, marker of brown adipose tissue, increased in Pdcd4 deficient PVAT of thoracic aorta in Apoe-/- mice after 19 weeks of high-fat diet. And Pdcd4 deficiency didn’t affect the expression of markers of brown adipose tissue and white adipose tissue such as UCP-1, aP2 and HOXC9. Furthermore, the morphology of PVAT was observed after H&E staining and Pdcd4 deficiency slightly attenuated white adipose tissue transformation in PVAT of thoracic aorta of Apoe-/- mice. These data suggested that PDCD4 may regulate the browning of PVAT.(4) PDCD4 had different effects on the inflammatory phenotype and browning of different depots of adipose tissue.In Apoe-/- mice after 19 weeks of high-fat diet, the regulatory roles of PDCD4 on the inflammatory phenotype and browning of different adipose depots were inconsistent. Pdcd4 deficiency decreased the level of IL-1β in both thoracic and abdominal PVAT. And in abdominal PVAT, Pdcd4 deficiency also increased the level of adiponectin meanwhile decreased the levels of MCP-1 and IFN-γ. Furthermore, in mesenteric adipose tissue, Pdcd4 deficiency down-regulated the expression of IL-6 and promoted the expression of PPAR-y and TGF-β. In addition, Pdcd4 deficiency significantly up-regulated the level of CIDEA, which is a marker of brown adipose tissue, in thoracic PVAT. Although Pdcd4 deficiency promoted the formation of anti-inflammatory phenotype and the browning of PVAT, however, in contrast with PVAT, PDCD4 had no effect on the inflammatory phenotype of visceral adipose tissue in Apoe-/- mice. These data demonstrated that PVAT was more sensitive to the regulation on inflammatory phenotype by PDCD4.(5) The effect of PDCD4 on PVAT of thoracic and abdominal aorta and PVAT related vascular diseases.We transplanted PVAT and epididymal adipose tissue from Apoe-/- and DKO mice into Apoe-/- mice. The graft was transplanted tightly adjacent to carotid artery of Apoe-/- mice. As a result, compared with the sham operation group, mice transplanted with Apoe-/- PVAT and Apoe-/- epididymal adipose tissue exhibited thickened arterial wall in carotid artery. However, mice transplanted with DKO PVAT and DKO epididymal adipose tissue had similar artery wall with sham operation group. Our results demonstrated that PDCD4 in PVAT affected adjacent artery and promoted vascular diseases.Conclusions1. PDCD4 plays a protective role in acute colitis and colitis-associated colorectal carcinoma via controlling IL-6/STAT3 pathway-mediated cell proliferation. Our research provided a new potential target for treating colitis and colitis-associated colorectal cancer by PDCD4.2. PDCD4 has distinct effect on the browning and inflammatory phenotype of perivascular adipose tissue surrounding thoracic and abdominal aorta, mesenteric adipose tissue, and epididymal adipose tissue, and thus regulates vascular diseases. Our research provided new insight to understand the inflammatory phenotype of perivascular adipose tissue and perivascular adipose tissue associated vascular disease, and indicated new target for clinical treatment.Originality and Significance1. We demonstrate, for the first time, that Pdcd4 deficiency promotes colonic epithelial cell proliferation via regulating IL-6/STAT3 pathway activation in mice, and thus accelerates colitis-associated colorectal carcinoma.2. We demonstrate, for the first time, that PDCD4 regulates browning and inflammatory phenotype of PVAT and thus affect vascular diseases. |
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