Smooth muscle alpha-actin expression and myofibroblast differentiation by TGFbeta are dependent upon MK2

Fibroblasts play a major role in processes such as wound repair, scarring, and fibrosis. Differentiation into myofibroblasts, characterized by upregulation of smooth muscle alpha-actin (smalpha) in response to profibrotic agents such as TGFbeta is believed to be an important step in fibrosis. Therefore, elucidating mechanisms of myofibroblast differentiation might reveal novel targets in treating diseases such as idiopathic pulmonary fibrosis (IPF). MK2 is a kinase substrate of p38 MAP kinase that mediates some effects of p38 activation on the actin cytoskeleton. Using mouse embryonic fibroblasts (MEF) from MK2 knockout (MK2(-/-)) mice, we demonstrate that disrupting expression of MK2 expression reduces filamentous actin and stress fibers. It also causes MK2(-/-) MEF to express less smalpha than their corresponding wild-type (WT) MEF at baseline and in response to TGFbeta. Furthermore, TGFbeta causes downregulation of smalpha in MK2(-/-) MEF, instead of upregulation observed in WT MEF. Expression of other fibroblast markers, such as collagen, is not altered in MK2(-/-) MEF. Our results further suggest that downregulation of smalpha in MK2(-/-) MEF is not due to lack of activation of serum responsive promoter elements, but probably due to reduced smalpha message stability in these cells. These results indicate that MK2 plays a key role in regulation of smalpha expression, and that targeting MK2 might present a therapeutic approach in managing conditions such as pulmonary fibrosis.     

Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine visceral leishmaniasis

Background

The immune response in the skin of dogs infected with Leishmania infantum is poorly understood, and limited studies have described the immunopathological profile with regard to distinct levels of tissue parasitism and the clinical progression of canine visceral leishmaniasis (CVL).

Methodology/Principal Findings

A detailed analysis of inflammatory cells (neutrophils, eosinophils, mast cells, lymphocytes, and macrophages) as well as the expression of chemokines (CCL2, CCL4, CCL5, CCL13, CCL17, CCL21, CCL24, and CXCL8) was carried out in dermis skin samples from 35 dogs that were naturally infected with L. infantum. The analysis was based on real-time polymerase chain reaction (PCR) in the context of skin parasitism and the clinical status of CVL. We demonstrated increased inflammatory infiltrate composed mainly of mononuclear cells in the skin of animals with severe forms of CVL and high parasite density. Analysis of the inflammatory cell profile of the skin revealed an increase in the number of macrophages and reductions in lymphocytes, eosinophils, and mast cells that correlated with clinical progression of the disease. Additionally, enhanced parasite density was correlated with an increase in macrophages and decreases in eosinophils and mast cells. The chemokine mRNA expression demonstrated that enhanced parasite density was positively correlated with the expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. In contrast, there was a negative correlation between parasite density and CCL24 expression.

Conclusions/Significance

These findings represent an advance in the knowledge about skin inflammatory infiltrates in CVL and the systemic consequences. Additionally, the findings may contribute to the design of new and more efficient prophylactic tools and immunological therapies against CVL.     

The expression of CCL2 by T lymphocytes of mammary tumor bearers: role of tumor-derived factors

Tumor-associated chemokines, including CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2), are thought to play many roles in cancer progression. Here we demonstrate the novel finding that during growth of the D1-7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is a dramatic up-regulation of CCL2 in splenic T cells at both the mRNA and protein levels upon stimulation. Of particular relevance is the finding that tumor-infiltrating T cells also produce high levels of CCL2. While a variety of tumor cell lines have been found to produce CCL2, we found no detectable levels of CCL2 protein in supernatants of the cultured mammary tumor cells. Investigation of the mechanisms involved in CCL2 induction showed that treatment of splenic T cells with the tumor-derived factors GM-CSF and phosphatidyl serine (PS) resulted in increased CCL2 production. This increased production may be involved in the downregulation of IFN-gamma by the T cells of tumor-bearing mice previously reported in this model, as treatment of splenic T lymphocytes with CCL2 resulted in a decreased secretion of IFN-gamma by those cells.     

Expression of the inflammatory chemokines CCL2, CCL5 and CXCL2 and the receptors CCR1-3 and CXCR2 in T lymphocytes from mammary tumor-bearing mice

Chemokines and their receptors have been studied in several solid tumor models as mediators of inflammation. In turn, inflammation has been implicated in the promotion and progression of tumors, and as such, chemokines have been proposed as novel molecular targets for chemotherapy. While the expression of these molecules has been described in tumor cells, endothelial cells, macrophages and neutrophils, less attention has been paid to the expression profile of these molecules by T lymphocytes in the periphery or infiltrating the tumor. Using the D1-DMBA-3 murine mammary adenocarcinoma model, we aimed to better characterize the differential expression of chemokines and/or their receptors in the host and in the tumor microenvironment, and specifically, in the T cells of tumor-bearing mice compared to normal control animals. We found that T lymphocytes from tumor-bearing mice express the pro-inflammatory chemokines, CCL2, CCL5 and CXCL2, as well as the chemokine receptors, CCR1, CCR2, CCR3 and CXCR2.     

CCL3-CCR5 axis regulates intratumoral accumulation of leukocytes and fibroblasts and promotes angiogenesis in murine lung metastasis process

Metastasis proceeds through interaction between cancer cells and resident cells such as leukocytes and fibroblasts. An i.v. injection of a mouse renal cell carcinoma, Renca, into wild-type mice resulted in multiple metastasis foci in lungs and was associated with intratumoral accumulation of macrophages, granulocytes, and fibroblasts. A chemokine, CCL3, was detected in infiltrating cells and, to a lesser degree, tumor cells, together with an infiltration of leukocytes expressing CCR5, a specific receptor for CCL3. A deficiency of the CCL3 or CCR5 gene markedly reduced the number of metastasis foci in the lung, and the analysis using bone marrow chimeric mice revealed that both bone marrow- and non-bone marrow-derived cells contributed to metastasis formation. CCL3- and CCR5-deficient mice exhibited a reduction in intratumoral accumulation of macrophages, granulocytes, and fibroblasts. Moreover, intratumoral neovascularization, an indispensable process for metastasis, was attenuated in these gene-deficient mice. Intrapulmonary expression of matrix metalloproteinase (MMP)-9 and hepatocyte growth factor (HGF) was enhanced in wild-type mice, and the increases were markedly diminished in CCL3- and CCR5-deficient mice. Furthermore, MMP-9 protein was detected in macrophages and granulocytes, the cells that also express CCR5 and in vitro stimulation by CCL3-induced macrophages to express MMP-9. Intratumoral fibroblasts expressed CCR5 and HGF protein. In vitro CCL3 stimulated fibroblasts to express HGF. Collectively, the CCL3-CCR5 axis appears to regulate intratumoral trafficking of leukocytes and fibroblasts, as well as MMP-9 and HGF expression, and as a consequence to accelerate neovascularization and subsequent metastasis formation.     

Cyclins of phases G1, S and G2/M are overexpressed in aneuploid mammary carcinomas

Expression of cyclins A, B1 and D1 in human breast cancer was analyzed using dual-parameter flow cytometry with simultaneous evaluation of the DNA content. The asynchronous MCF-7 breast adenocarcinoma cells were used to implement flow cytometry analysis and to analyze the cell cycle distribution of cyclins. The patterns of the cyclin expression were also analyzed in vivo in fresh tissue specimens of human breast carcinomas. The combined measurement of DNA and cyclins showed a higher cyclin expression in aneuploid (11.5 +/- 2.0%, 4.3 +/- 1.1%, and 19.5 +/- 3.4% positive cells for cyclins A, B, and D1, respectively) than in diploid carcinomas (3.9 +/- 1.2%, 1.1 +/- 0.4%, and 5.0 +/- 1.2% positive cells for cyclins A, B, and D1, respectively). A positive relationship was also found between cyclin A and D1 expression and H(3)-thymidine labeling index. In the in vitro model, the asynchronous growing MCF-7 cells showed a variable number of cells expressing cyclins in an unscheduled way, unrelated to the phase at which these cyclins are expressed in normal cells. A similar condition was also observed in tumors. In conclusion, the data showed a deregulated expression of cyclins in a transformed adenocarcinoma cell line and in breast tumors. Furthermore, overexpression of these proteins is related to the aneuploid and high proliferative activity of human mammary carcinomas.     

Pro-inflammatory cytokines IL-6 and CCL2 suppress expression of circadian gene Period2 in mammary epithelial cells

Chronic inflammation is known to contribute to tumor initiation and cancer progression. In breast tissue, the core circadian gene Period (PER)2 plays a critical role in mammary gland development and possesses tumor suppressor function. Interleukin (IL)-6 and C-C motif chemokine ligand (CCL) 2 are among the most abundant cytokines in the inflammatory microenvironment. We found that acute stimulation by IL-6/CCL2 reduced PER2 expression in non-tumorigenic breast epithelial cells. Longer term exposure to IL-6/CCL2 suppressed PER2 to an even lower level. IL-6 activated STAT3/NFκB p50 signaling to recruit HDAC1 to the PER2 promoter. CCL2 activated the PI3K/AKT pathway to promote ELK-1 cytoplasm-to-nucleus translocation, recruit HDAC1 to the proximal PER2 promoter and facilitate DNMT3-EZH2-PER2 promoter association. Ectopic expression of PER2 inhibited IL-6 or CCL2 induced mammosphere forming ability and reduced sphere size indicating that PER2 repression in breast epithelial cells can be crucial to activate tumorigenesis in an inflammatory microenvironment. The diminished expression of PER2 can be observed over a time scale of hours to weeks following IL-6/CCL2 stimulation suggesting that PER2 suppression occurs in the early stage of the interaction between an inflammatory microenvironment and normal breast epithelial cells. These data show new mechanisms by which mammary cells interact with a cancerous microenvironment and provide additional evidence that PER2 expression contributes to breast tumorigenesis.     

Up-regulated expression of Ezrin and c-Met proteins are related to the metastasis and prognosis of gastric carcinomas

Recent publications demonstrated that abnormal expression of Ezrin and c-Met proteins were related to carcinogenesis, metastasis and prognosis of various sorts of tumors. In this study we detected the expressions of Ezrin and c-Met proteins in normal gastric mucosa, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinoma and analyzed the correlations with metastasis and prognosis of gastric carcinomas. The results demonstrated that both Ezrin and c-Met overexpression were related to the occurrence and progression of gastric carcinoma. Our findings also demonstrated that combined detection of these two tumor-specific biomarkers in gastric carcinomas can provide additional efficacy in predicting the patients' outcomes.     

Proinflammatory cytokine production in liver regeneration is Myd88-dependent, but independent of Cd14, Tlr2, and Tlr4

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.     

Growth inhibition of murine mammary carcinoma by monoclonal IgE antibodies specific for the mammary tumor virus

Summary Two IgE-producing hybridomas were established from spleen cells of Balb/c mice, which had been immunized with mouse mammary tumor virus (MMTV). These IgE monoclonal antibodies (mAbs) reacted specifically with the major envelope glycoprotein (gp36) of MMTV, as established by the immunoblot assay and by passive cutaneous anaphylaxis. The effect of the IgE mAbs (produced by clone A8) on the growth of the MMTV-secreting mammary adenocarcinoma H2712 was investigated in syngeneic C3H/HeJ mice. The mice were inoculated s.c. with either 10⁵ (100 × LD₅₀) or 10⁶ (1000 × LD₅₀) tumor cells and received repeated i.p. injections of 25 µg anti-gp36 IgE mAbs at 4-day intervals for 8 weeks. This treatment prevented the development of subcutaneous tumors in 50% of the animals. Similar protection was observed when the tumor cells (10⁵/animal) were injected i.p. 4 days prior to the beginning of the i.p. treatment consisting of injections of 25 µg mAbs at 4-day intervals for 6 weeks. However, these mAbs did not protect C3H/HeJ mice against the MMTV-negative MA16/c carcinoma cells. Hence, these results support the view that IgE-mediated cytotoxic mechanisms may play an immunologically specific antitumor surveillance role and that laboratory-induced antitumor IgE mAbs have the potential of specific therapeutic agents for in vivo destruction of tumor cells.     

草鱼MyD88基因组结构解析及启动子活性探讨

{{@ convertAbstractHtml(article.abstractinfoCn, "cn")}}       

Initial stages of neural regeneration in Helisoma trivolvis are dependent upon PLA2 activity

Neuronal regeneration after damage to an axon tract requires the rapid sealing of the injured plasma membrane and the subsequent formation of growth cones that can lead regenerating processes to their appropriate target. Membrane sealing and growth cone formation are Ca(2+)-dependent processes, but the signaling pathways activated by Ca(2+) to bring about these effects remain poorly understood. An in vitro injury model was employed in which neurites from identified snail neurons (Helisoma trivolvis) were transected with a glass microknife, and the formation of new growth cones from the distal portions of transected neurites was recorded at defined times after transection. This study presents three main results. First, phospholipase A(2) (PLA(2)), a calcium-activated enzyme, is necessary for membrane sealing in vitro. Second, PLA(2) activity is also required for the formation of a new growth cone after the membrane has sealed successfully. Thus, PLA(2) plays a dual role by affecting both growth cone formation and membrane sealing. Third, the injury-induced activation of PLA(2) by Ca(2+) controls growth cone formation through the production of leukotrienes, secondary metabolites of PLA(2) activity. Taken together, these results suggest that the injury-induced Ca(2+) influx acts via PLA(2) and leukotriene production to assure growth cone formation. These findings indicate that events that cause an inhibition of PLA(2) or lipoxygenases, enzymes that produce leukotrienes, could result in the inability of neurites to regenerate.     

CCL2 and CCL3 are essential mediators of pelvic pain in experimental autoimmune prostatitis

Experimental autoimmune prostatitis (EAP) is a murine model of chronic prostatitis/chronic pelvic pain syndrome (CPPS) in men, a syndrome characterized by chronic pelvic pain. We have demonstrated that chemokine ligands CCL2 and CCL3 are biomarkers that correlate with pelvic pain symptoms. We postulated that CCL2 and CCL3 play a functional role in CPPS and therefore examined their expression in EAP. Upon examination of the prostate 5 days after induction of EAP, CCL2 mRNA was elevated 2- to 3-fold, CCL8 by 15-fold, CCL12 by 12- to 13-fold, and CXCL9 by 2- to 4-fold compared with control mice. At 10 days the major chemokines were CXCL13 and CXCL2; at 20 days CCL2 (1- to 2-fold), CCL3 (2- to 3-fold) and CCL11 (2- to 3-fold); and at 30 days, CCL12 (20- to 35-fold) and smaller increases in CCL2, CCL3, and XCL1. Chemokine elevations were accompanied by increases in mast cells and B cells at 5 days, monocytes and neutrophils at day 10, CD4+ T cells at day 20, and CD4+ and CD8+ T cells at day 30. Anti-CCL2 and anti-CCL3 neutralizing antibodies administered at EAP onset attenuated pelvic pain development, but only anti-CCL2 antibodies were effective therapeutically. CCL2- and its cognate receptor CCR2-deficient mice were completely protected from development of pain symptoms but assumed susceptibility after reconstitution with wild-type bone marrow. CCL3-deficient mice showed resistance to the maintenance of pelvic pain while CCR5-deficient mice did not show any lessening of pelvic pain severity. These results suggest that the CCL2-CCR2 axis and CCL3 are important mediators of chronic pelvic pain in EAP.