Matrix metalloproteinase-8 (MMP-8) regulates the activation of hepatic stellate cells (HSCs) through the ERK-mediated pathway.

Hepatic stellate cells (HSCs) are known to play a key role in the progression of liver fibrosis by producing excessive extracellular matrix (ECM). Matrix metalloproteinases (MMPs) belong to a family of endopeptidases, which have a well-established role in the degradation of ECM. Our study suggests that, besides the degradation of the extracellular matrix, matrix metalloproteinase-8 (MMP-8) has a non-canonical role in activating the quiescent HSCs to myofibroblasts by regulating the expression of Col1A1 and αSMA. We have identified that MMP-8 secreted from macrophages as a response to LPS stimulation activates HSCs via ERK1/2-dependent pathway. In addition to this, we determined that MMP-8 may regulate the homodimerization of c-Jun in LX-2 cells, during the trans-differentiation process from quiescent HSC to activate myofibroblasts. Macrophage-released MMP-8 plays a master role in activating the dormant HSCs to activate myofibroblasts through the Erk-mediated pathway and Jun cellular translocation leading to liver fibrosis. Significance MMP-8 can be used as a therapeutic target against liver fibrosis.

The TLR4-NOS1-AP1 signaling axis regulates macrophage polarization.

OBJECTIVE: Macrophages polarize to proinflammatory M1 or anti-inflammatory M2 states with distinct physiological functions. This transition within the M1-M2 phenotypes decides the nature, duration and severity of an inflammatory response. Although there is a substantial understanding of the fate of these phenotypes, the underlying molecular mechanism of transition within the M1-M2 phenotypes is not well understood. We have investigated the role of neuronal nitric oxide synthase (NOS1)-mediated regulation of activator protein 1 (AP-1) transcription factor in macrophages as a critical effector of macrophage phenotypic change. MATERIALS AND METHODS: Raw 264.7 and THP1 macrophages were stimulated with LPS (250 ng/ml) to activate the inflammatory signaling pathway. We analyzed the effect of pharmacological NOS1 inhibitor: TRIM (1-(2- Trifluoromethylphenyl) imidazole) on LPS-induced inflammatory response in macrophages. RESULTS: We determined that NOS1-derived nitric oxide (NO) facilitate Fos and Jun interaction which induces IL-12 & IL-23 expression. Pharmacological inhibition of NOS1 inhibits ATF2 and Jun dimer. Switching of Fos and Jun dimer to ATF2 and Jun dimerization controls phenotype transition from IL-12high IL-23high IL-10low to IL-12low IL-23lowIL-10high phenotype, respectively. CONCLUSION: These findings highlight a key role of the TLR4-NOS1-AP1 signaling axis in regulating macrophage polarization.

Male Gonads Transplantation from Kadaknath Chicken to Chicken and Duck Surrogates.

Transplantation of the gonadal tissue of male and female avian species, such as chicken, onto suitable surrogates and production of live offspring has been successfully demonstrated as a strategy for the conservation and re-constitution of valuable chicken germplasm. The main objective of this study was to establish and develop the male gonadal tissue transplantation technology for the conservation of the indigenous chicken germplasm. The male gonads of the Indian native chicken breed, Kadaknath (KN), were transplanted from a day-old donor to a recipient white leghorn (WL) chicken, and Khaki Campbell (KC) ducks, as surrogates. All the surgical interventions were performed under permitted general anaesthesia, and the chicks, upon recovery, were reared with and without immunosuppressant. The recipient surrogates for the donor KN gonads were housed and reared for 10-14 weeks, and post-sacrifice, developed gonadal tissues were harvested to squeeze out the fluid to perform artificial insemination (AI). The AI-entailed fertility test using the recovered seminal extract from the transplanted KN testes from both surrogate species (KC ducks and WL males), used against KN purebred females, remained very close to the percent fertility realised from purebred KN chickens (controls). These initial results revealed from this trial study suggest definitively that, Kadaknath male gonads were readily accepted and grown inside the intra- and inter-species surrogate host, WL chicken and KC ducks, demonstrating a suitable intra- and inter-species donor-host system. Furthermore, the developed transplanted male gonads of KN chicken into the surrogates were found to have the potential to fertilise the egg and give rise to pure-line KN chicks.

Repurposing Thioridazine (TDZ) as an anti-inflammatory agent.

Nuclear factor-kB (NF-kB) is a crucial transcription factor in the signal transduction cascade of the inflammatory signaling. Activation of NF-κB depends on the phosphorylation of IκBα by IκB kinase (IKKß) followed by subsequent ubiquitination and degradation. This leads to the nuclear translocation of the p50- p65 subunits of NF-κB, and further triggers pro-inflammatory cytokine gene expression. Thus, in the need of a more effective therapy for the treatment of inflammatory diseases, specific inhibition of IKKß represents a rational alternative strategy to the current therapies. A computer-aided drug identification protocol was followed to identify novel IKKß inhibitors from a database of over 1500 Food and Drug Administration (FDA) drugs. The best scoring compounds were compared with the already known high-potency IKKß inhibitors for their ability to bind and inhibit IKKß by evaluating their docking energy. Finally, Thioridazinehydrochloride (TDZ), a potent antipsychotic drug against Schizophrenia was selected and its efficiency in inhibiting IκBα protein degradation and NF-κB activation was experimentally validated. Our study has demonstrated that TDZ blocks IκBα protein degradation and subsequent NF-κB activation to inhibit inflammation. Thus, it is a potential repurposed drug against inflammation.

Matrix Metalloproteinases (MMPs) in Liver Diseases.

Matrix metalloproteinases (MMPs) are proteinases capable of degrading components of the extracellular matrix and numerous nonmatrix proteins. MMPs along with tissue inhibitors of MMPs, have been implicated in the pathogenesis of liver diseases. Although, the precise mechanism-of-actions of MMPs in various liver related disorders is largely unknown, however, data from diverse experimental models indicate that these proteinases influence cellular activities including proliferation and survival, gene expression, as well as multiple aspects of inflammation. Hence, MMP's are likely key players in the outcomes related to liver disease.

Heterotrimeric complex of p38 MAPK, PKCδ, and TIRAP is required for AP1 mediated inflammatory response.

Inflammation could be described as a physiological response of the body to tissue injury, pathogen invasion, and irritants. During the inflammatory phase, cells of both the innate as well as adaptive immune system are activated and recruited to the site of inflammation. These mediators are downstream targets for the transcription factors; activator protein-1 (AP1), nuclear factor kappa-light-chain-enhancer (NF-κB), signal transducers and activators of transcription factors (STAT1), as well as interferon regulatory factors (IRFs), which control the expression of most immunomodulatory genes. There is a significant increase in active p38 mitogen-activated protein kinase (p38MAK) immediately after lipopolysaccharide (LPS) stimulation, which results in the activation of AP-1 transcription factor and expression of proinflammatory cytokines, IL-12 and IL-23. We studied the novel mechanism of p38 MAPK activation through the formation of a heterotrimeric complex of Protein kinase C delta type (PKCδ), Toll-Interleukin 1 Receptor (TIR) Domain Containing Adaptor Protein (TIRAP), and p38 proteins. TIRAP serves as an adaptor molecule which brings PKCδ and p38 in close proximity. The complex facilitates the activation of p38MAPK by PKCδ. Therefore, we propose that disruption of the heterotrimeric complex may be a good strategy to dampen the inflammatory response. Structure-based design of small molecules or peptides targetting PKCδ-TIRAP or TIRAP-p38 interfaces would be beneficial for therapy in AP1 mediated inflammatory diseases.

Argonaute-2-null embryonic stem cells are retarded in self-renewal and differentiation.

RNA interference (RNAi) pathways regulate self-renewal and differentiation of embryonic stem (ES) cells. Argonaute 2 (Ago2) is a vital component of RNA-induced silencing complex (RISC) and the only Ago protein with slicer activity. We generated Ago2-deficient ES cells by conditional gene targeting. Ago2-deficient ES cells are defective in the small-RNA-mediated gene silencing and are significantly compromised in biogenesis of mature microRNA. The self-renewal rate of Ago2-deficient ES cells is affected due to failure of silencing of Cdkn1a by EScell- specific microRNAs (miRNA) in the absence of Ago2. Interestingly, unlike Dicer- and Dgcr8-deficient ES cells, they differentiate to all three germ layers both in vivo and in vitro. However, early differentiation of Ago2-deficient ES cells is delayed by 2-4 days as indicated by persistence of higher levels of self-renewal/ pluripotency markers during differentiation. Further, appearance of morphological and differentiation markers is also delayed during the differentiation. In this study we show that Ago2 is essential for normal self-renewal and differentiation. Also, our data suggest that self-renewal and differentiation of ES cells are regulated by both siRNA and miRNA pathways.

Argonaute-2-null embryonic stem cells are retarded in self-renewal and differentiation.

RNA interference (RNAi) pathways regulate self-renewal and differentiation of embryonic stem (ES) cells. Argonaute 2 (Ago2) is a vital component of RNA-induced silencing complex (RISC) and the only Ago protein with slicer activity. We generated Ago2-deficient ES cells by conditional gene targeting. Ago2-deficient ES cells are defective in the small-RNA-mediated gene silencing and are significantly compromised in biogenesis of mature microRNA. The self-renewal rate of Ago2-deficient ES cells is affected due to failure of silencing of Cdkn1a by EScell- specific microRNAs (miRNA) in the absence of Ago2. Interestingly, unlike Dicer- and Dgcr8-deficient ES cells, they differentiate to all three germ layers both in vivo and in vitro. However, early differentiation of Ago2-deficient ES cells is delayed by 2–4 days as indicated by persistence of higher levels of self-renewal/ pluripotency markers during differentiation. Further, appearance of morphological and differentiation markers is also delayed during the differentiation. In this study we show that Ago2 is essential for normal self-renewal and differentiation. Also, our data suggest that self-renewal and differentiation of ES cells are regulated by both siRNA and miRNA pathways.