Amelioration of Fibrosis via S1P Inhibition Is Regulated by Inactivation of TGF-ß and SPL Pathways in the Human Cornea.

Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to both, transforming growth factor beta (TGF-ß) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-ß and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-ß1 (ß1), 1 µM sphingosine-1-phosphate (S1P), and 5 µM Sphingosine kinase inhibitor 2 (I2). Five groups were tested: (1) control (no treatment); rescue groups; (2) ß1/S1P; (3) ß1/I2; prevention groups; (4) S1P/ß1; and (5) I2/ß1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-ß signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-ß binding proteins (LTBPs), TGF-ß receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I2 prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I2 treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-ß receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I2 as a novel therapy for corneal fibrosis.

Sphingolipid biosynthetic inhibitor L-Cycloserine prevents oxidative-stress-mediated death in an in vitro model of photoreceptor-derived 661W cells.

Oxidative stress plays a pivotal role in the pathogenesis of several neurodegenerative diseases. Retinal degeneration causes irreversible death of photoreceptor cells, ultimately leading to vision loss. Under oxidative stress, the synthesis of bioactive sphingolipid ceramide increases, triggering apoptosis in photoreceptor cells and leading to their death. This study investigates the effect of L-Cycloserine, a small molecule inhibitor of ceramide biosynthesis, on sphingolipid metabolism and the protection of photoreceptor-derived 661W cells from oxidative stress. The results demonstrate that treatment with L-Cycloserine, an inhibitor of Serine palmitoyl transferase (SPT), markedly decreases bioactive ceramide and associated sphingolipids in 661W cells. A nontoxic dose of L-Cycloserine can provide substantial protection of 661W cells against H2O2-induced oxidative stress by reversing the increase in ceramide level observed under oxidative stress conditions. Analysis of various antioxidant, apoptotic and sphingolipid pathway genes and proteins also confirms the ability of L-Cycloserine to modulate these pathways. Our findings elucidate the generation of sphingolipid mediators of cell death in retinal cells under oxidative stress and the potential of L-Cycloserine as a therapeutic candidate for targeting ceramide-induced degenerative diseases by inhibiting SPT. The promising therapeutic prospect identified in our findings lays the groundwork for further validation in in-vivo and preclinical models of retinal degeneration.

Mouse Model of Nitrogen Mustard Ocular Surface Injury Characterization and Sphingolipid Signaling.

Vesicating chemicals like sulfur mustard (SM) or nitrogen mustard (NM) can cause devastating damage to the eyes, skin, and lungs. Eyes, being the most sensitive, have complicated pathologies that can manifest immediately after exposure (acute) and last for years (chronic). No FDA-approved drug is available to be used as medical counter measures (MCMs) against such injuries. Understanding the pathological mechanisms in acute and chronic response of the eye is essential for developing effective MCMs. Here, we report the clinical and histopathological characterization of a mouse model of NM-induced ocular surface injury (entire surface) developed by treating the eye with 2% (w/v) NM solution for 5 min. Unlike the existing models of specific injury, our model showed severe ocular inflammation, including the eyelids, structural deformity of the corneal epithelium and stroma, and diminished visual and retinal functions. We also observed alterations of the inflammatory markers and their expression at different phases of the injury, along with an activation of acidic sphingomyelinase (aSMase), causing an increase in bioactive sphingolipid ceramide and a reduction in sphingomyelin levels. This novel ocular surface mouse model recapitulated the injuries reported in human, rabbit, and murine SM or NM injury models. NM exposure of the entire ocular surface in mice, which is similar to accidental or deliberate exposure in humans, showed severe ocular inflammation and caused irreversible alterations to the corneal structure and significant vision loss. It also showed an intricate interplay between inflammatory markers over the injury period and alteration in sphingolipid homeostasis in the early acute phase.

Potentiation of Sphingolipids and TGF-ß in the human corneal stroma reveals intricate signaling pathway crosstalks.

Corneal haze brought on by fibrosis due to insult can lead to partial or complete vision loss. Currently, corneal transplantation is the gold standard for treating severe corneal fibrosis, which comes with the risk of rejection and the issue of donor tissue shortages. Sphingolipids (SPLs) are known to be associated with fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to Transforming Growth Factor ß (TGF-ß) signaling and corneal fibrogenesis. This study aimed to elucidate the interplay of SPLs, specifically sphingosine-1-phosphate (S1P) signaling, and its' interactions with TGF-ß signaling through detailed analyses of the corresponding downstream signaling targets in the context of corneal fibrosis, in vitro. Healthy human corneal fibroblasts (HCFs) were isolated, plated on polycarbonate membranes, and stimulated with a stable Vitamin C derivative. The 3D constructs were treated with either 5 µM sphingosine-1-phosphate (S1P), 5 µM SPHK I2 (I2; inhibitor of sphingosine kinase 1, one of the two enzymes responsible for generating S1P in mammalian cells), 0.1 ng/mL TGF-ß1, or 0.1 ng/mL TGF-ß3. Cultures with control medium-only served as controls. All 3D constructs were examined for protein expression of fibrotic markers, SPLs, TGF-ßs, and relevant downstream signaling pathways. This data revealed no significant changes in any LTBP (latent TGF-ß binding proteins) expression when stimulated with S1P or I2. However, LTBP1 was significantly upregulated via stimulation of TGF-ß1 and TGF-ß3, whereas LTBP2 was significantly upregulated only with TGF-ß3 stimulation. Significant downregulation of TGF-ß receptor II (TGF-ßRII) following S1P stimulation but significant upregulation following I2 stimulation was observed. Following TGF-ß1, S1P, and I2 stimulation, phospho-SMAD2 (pSMAD2) was significantly downregulated. Furthermore, I2 stimulation led to significant downregulation of SMAD4. Adhesion/proliferation/transcription regulation targets, SRC, FAK, and pERK 1/2 were all significantly downregulated by exogenous S1P, whereas I2 only significantly downregulated FAK. Exogenous TGF-ß3 caused significant upregulation of AKT. Interestingly, both I2 and TGF-ß3 caused significant downregulation of JNK expression. Lastly, TGF-ß1 led to significant upregulation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1PR3), whereas TGF-ß3 caused significant upregulation of only SphK1. Together with previously published work from our group and others, S1P inhibition exhibits great potential as an efficacious anti-fibrotic modality in human corneal stromal ECM. The current findings shed further light on a very complex and rather incompletely investigated mechanism, and cement the intricate crosstalk between SPLs and TGF-ß in corneal fibrogenesis. Future studies will dictate the potential of utilizing SPLs/TGF-ß signaling modulators as novel therapeutics in corneal fibrosis.

Angiogenesis Model of Cornea to Understand the Role of Sphingosine 1-Phosphate.

The bioactive sphingolipid sphingosine 1-phosphate (S1P) and its five cognate receptors (S1PR1-5) have been implicated to play important role in multiple aspects of human physiology and diseases. The S1P-S1PR1 signaling axis in endothelial cells is crucial for establishing flow competent blood vessels. The role of S1P in neovascular pathology is of great interest and is evolving as a promising target for treatment. Here we describe an easy and affordable in vivo model of corneal neovascularization using an alkali chemical burn to the cornea. This method gives a consistent and easy-to-quantitate procedure for neovascularization and angiogenesis studies.

Image-Based Longitudinal Characterization of Corneal Wound to Understand the Role of Sphingosine-1-Phosphate.

Since its discovery, the bioactive sphingolipid sphingosine 1-phosphate (S1P) has been shown to involve in a myriad of cellular and physiological processes. In the process of tissue healing, S1P plays an important role in both normal and pathological healing, leading to fibrosis in multiple tissues including the cornea. Cornea covers the anterior portion of the eye and is responsible for the refraction of light. Corneal transparency is essential to obtain a clear vision, and a proper wound healing process is necessary for a clear cornea. Even though S1P is indicated to be a critical player in corneal fibrosis, we lack a detailed understanding of the role of S1P signaling in corneal wound healing and fibrosis. Herein, we describe a methodology to characterize the in-vivo wound healing process of the cornea using an easy and affordable imaging-based assay. This gives a consistent and easy way to characterize the wound and also the longitudinal healing process.

Ablation of Sphingosine Kinase 1 Protects Cornea from Neovascularization in a Mouse Corneal Injury Model.

The purpose of this study was to investigate the role of sphingosine kinase 1 (SphK1), which generates sphingosine-1-phosphate (S1P), in corneal neovascularization (NV). Wild-type (WT) and Sphk1 knockout (Sphk1-/-) mice received corneal alkali-burn treatment to induce corneal NV by placing a 2 mm round piece of Whatman No. 1 filter paper soaked in 1N NaOH on the center of the cornea for 20 s. Corneal sphingolipid species were extracted and identified using liquid chromatography/mass spectrometry (LC/MS). The total number of tip cells and those positive for ethynyl deoxy uridine (EdU) were quantified. Immunocytochemistry was done to examine whether pericytes were present on newly forming blood vessels. Cytokine signaling and angiogenic markers were compared between the two groups using multiplex assays. Data were analyzed using appropriate statistical tests. Here, we show that ablation of SphK1 can significantly reduce NV invasion in the cornea following injury. Corneal sphingolipid analysis showed that total levels of ceramides, monohexosyl ceramides (HexCer), and sphingomyelin were significantly elevated in Sphk-/- corneas compared to WT corneas, with a comparable level of sphingosine among the two genotypes. The numbers of total and proliferating endothelial tip cells were also lower in the Sphk1-/- corneas following injury. This study underscores the role of S1P in post-injury corneal NV and raises further questions about the roles played by ceramide, HexCer, and sphingomyelin in regulating corneal NV. Further studies are needed to unravel the role played by bioactive sphingolipids in maintenance of corneal transparency and clear vision.

Hydroxychloroquine Causes Early Inner Retinal Toxicity and Affects Autophagosome-Lysosomal Pathway and Sphingolipid Metabolism in the Retina.

Hydroxychloroquine (HCQ) is an anti-malarial drug but also widely used to treat autoimmune diseases like arthritis and lupus. Although there have been multiple reports of the adverse effect of prolonged HCQ usage on the outer retina, leading to bull's-eye maculopathy, the effect of HCQ toxicity on the inner retina as well as on overall visual functions has not been explored in detail. Furthermore, lack of an established animal model of HCQ toxicity hinders our understanding of the underlying molecular mechanisms. Here, using a small clinical study, we confirmed the effect of HCQ toxicity on the inner retina, in particular the reduction in central inner retinal thickness, and established a mouse model of chronic HCQ toxicity that recapitulates the effects observed in human retina. Using the mouse model, we demonstrated that chronic HCQ toxicity results in loss of inner retinal neurons and retinal ganglion cells (RGC) and compromises visual functions. We further established that HCQ treatment prevents autophagosome-lysosome fusion and alters the sphingolipid homeostasis in mouse retina. Our results affirm the notion that HCQ treatment causes early damage to the inner retina and affects visual functions before leading to characteristic toxicity in the macular region of the outer retina, 'bull's-eye maculopathy.' We also provide insights into the underlying molecular mechanisms of HCQ retinal toxicity that may involve autophagy-lysosomal defects and alterations in sphingolipid metabolism.

Sphingolipids as critical players in retinal physiology and pathology.

Sphingolipids have emerged as bioactive lipids involved in the regulation of many physiological and pathological processes. In the retina, they have been established to participate in numerous processes, such as neuronal survival and death, proliferation and migration of neuronal and vascular cells, inflammation, and neovascularization. Dysregulation of sphingolipids is therefore crucial in the onset and progression of retinal diseases. This review examines the involvement of sphingolipids in retinal physiology and diseases. Ceramide (Cer) has emerged as a common mediator of inflammation and death of neuronal and retinal pigment epithelium cells in animal models of retinopathies such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa. Sphingosine-1-phosphate (S1P) has opposite roles, preventing photoreceptor and ganglion cell degeneration but also promoting inflammation, fibrosis, and neovascularization in AMD, glaucoma, and pro-fibrotic disorders. Alterations in Cer, S1P, and ceramide 1-phosphate may also contribute to uveitis. Notably, use of inhibitors that either prevent Cer increase or modulate S1P signaling, such as Myriocin, desipramine, and Fingolimod (FTY720), preserves neuronal viability and retinal function. These findings underscore the relevance of alterations in the sphingolipid metabolic network in the etiology of multiple retinopathies and highlight the potential of modulating their metabolism for the design of novel therapeutic approaches.

Role of ceramides in the pathogenesis of diabetes mellitus and its complications.

Diabetes mellitus (DM) is a systemic metabolic disease that affects 463 million adults worldwide and is a leading cause of cardiovascular disease, blindness, nephropathy, peripheral neuropathy, and lower-limb amputation. Lipids have long been recognized as contributors to the pathogenesis and pathophysiology of DM and its complications, but recent discoveries have highlighted ceramides, a class of bioactive sphingolipids with cell signaling and second messenger capabilities, as particularly important contributors to insulin resistance and the underlying mechanisms of DM complications. Besides their association with insulin resistance and pathophysiology of type 2 diabetes, evidence is emerging that certain species of ceramides are mediators of cellular mechanisms involved in the initiation and progression of microvascular and macrovascular complications of DM. Advances in our understanding of these associations provide unique opportunities for exploring ceramide species as potential novel therapeutic targets and biomarkers. This review discusses the links between ceramides and the pathogenesis of DM and diabetic complications and identifies opportunities for novel discoveries and applications.

A RAS-CaMKKß-AMPKα2 pathway promotes senescence by licensing post-translational activation of C/EBPß through a novel 3'UTR mechanism.

Oncogene-induced senescence (OIS) is an intrinsic tumor suppression mechanism that requires the p53 and RB pathways and post-translational activation of C/EBPß through the RAS-ERK cascade. We previously reported that in transformed/proliferating cells, C/EBPß activation is inhibited by G/U-rich elements (GREs) in its 3'UTR. This mechanism, termed "3'UTR regulation of protein activity" (UPA), maintains C/EBPß in a low-activity state in tumor cells and thus facilitates senescence bypass. Here we show that C/EBPß UPA is overridden by AMPK signaling. AMPK activators decrease cytoplasmic levels of the GRE binding protein HuR, which is a key UPA component. Reduced cytoplasmic HuR disrupts 3'UTR-mediated trafficking of Cebpb transcripts to the peripheral cytoplasm-a fundamental feature of UPA-thereby stimulating C/EBPß activation and growth arrest. In primary cells, oncogenic RAS triggers a Ca++-CaMKKß-AMPKα2-HuR pathway, independent of AMPKα1, that is essential for C/EBPß activation and OIS. This axis is disrupted in cancer cells through down-regulation of AMPKα2 and CaMKKß. Thus, CaMKKß-AMPKα2 signaling constitutes a key tumor suppressor pathway that activates a novel UPA-cancelling mechanism to unmask the cytostatic and pro-senescence functions of C/EBPß.

Oncogenic RAS-Induced Perinuclear Signaling Complexes Requiring KSR1 Regulate Signal Transmission to Downstream Targets.

The precise characteristics that distinguish normal and oncogenic RAS signaling remain obscure. Here, we show that oncogenic RAS and BRAF induce perinuclear relocalization of several RAS pathway proteins, including the kinases CK2 and p-ERK1/2 and the signaling scaffold KSR1. This spatial reorganization requires endocytosis, the kinase activities of MEK-ERK and CK2, and the presence of KSR1. CK2α colocalizes with KSR1 and Rab11, a marker of recycling endosomes, whereas p-ERK associates predominantly with a distinct KSR1-positive endosomal population. Notably, these perinuclear signaling complexes (PSC) are present in tumor cell lines, mouse lung tumors, and mouse embryonic fibroblasts undergoing RAS-induced senescence. PSCs are also transiently induced by growth factors (GF) in nontransformed cells with delayed kinetics (4-6 hours), establishing a novel late phase of GF signaling that appears to be constitutively activated in tumor cells. PSCs provide an essential platform for RAS-induced phosphorylation and activation of the prosenescence transcription factor C/EBPß in primary MEFs undergoing senescence. Conversely, in tumor cells, C/EBPß activation is suppressed by 3'UTR-mediated localization of Cebpb transcripts to a peripheral cytoplasmic domain distinct from the PSC region. Collectively, our findings indicate that sustained PSC formation is a critical feature of oncogenic RAS/BRAF signaling in cancer cells that controls signal transmission to downstream targets by regulating selective access of effector kinases to substrates such as C/EBPß.Significance: In addressing the long-standing question of the difference between normal and oncogenic RAS pathway signaling, this study shows that oncogenic RAS specifically triggers constitutive endocytosis-dependent movement of effector kinases to a perinuclear region, thereby creating connections to unique downstream targets such as the core prosenescence and the inflammatory regulatory transcription factor C/EBPß. Cancer Res; 78(4); 891-908. ©2017 AACR.

Identification of senescent cell surface targetable protein DPP4.

Senescent cell accumulation in aging tissues is linked to age-associated diseases and declining function, prompting efforts to eliminate them. Mass spectrometry analysis revealed that DPP4 (dipeptidyl peptidase 4) was selectively expressed on the surface of senescent, but not proliferating, human diploid fibroblasts. Importantly, the differential presence of DPP4 allowed flow cytometry-mediated isolation of senescent cells using anti-DPP4 antibodies. Moreover, antibody-dependent cell-mediated cytotoxicity (ADCC) assays revealed that the cell surface DPP4 preferentially sensitized senescent, but not dividing, fibroblasts to cytotoxicity by natural killer cells. In sum, the selective expression of DPP4 on the surface of senescent cells enables their preferential elimination.

3'UTR elements inhibit Ras-induced C/EBPß post-translational activation and senescence in tumour cells.

C/EBPß is an auto-repressed protein that becomes post-translationally activated by Ras-MEK-ERK signalling. C/EBPß is required for oncogene-induced senescence (OIS) of primary fibroblasts, but also displays pro-oncogenic functions in many tumour cells. Here, we show that C/EBPß activation by H-Ras(V12) is suppressed in immortalized/transformed cells, but not in primary cells, by its 3' untranslated region (3'UTR). 3'UTR sequences inhibited Ras-induced cytostatic activity of C/EBPß, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3'UTR suppressed induction of senescence-associated C/EBPß target genes, while promoting expression of genes linked to cancers and TGFß signalling. An AU-rich element (ARE) and its cognate RNA-binding protein, HuR, were required for 3'UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPß translation controls de-repression by Ras signalling. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras-induced C/EBPß activation and OIS to proceed. Our findings reveal a novel mechanism whereby non-coding mRNA sequences selectively regulate C/EBPß activity and suppress its anti-oncogenic functions.

Leishmania requires Rab7-mediated degradation of endocytosed hemoglobin for their growth.

Leishmania is unable to synthesize heme and must acquire it from exogenous source, the mechanism of which is not known. We have shown that Leishmania endocytoses hemoglobin (Hb) and subsequently degrade it probably to generate heme. To understand how internalized Hb is degraded, we have cloned and expressed Rab7 homolog from Leishmania donovani. Interestingly, Rab7 in Leishmania is found to be localized both on early and late endocytic compartment and regulates both uptake and degradation of endocytosed Hb demonstrating that Rab7 in Leishmania play a very unique role connecting both early and late events of Hb endocytosis. Our data also indicate that overexpression of Rab7:WT in Leishmania induces transport of Hb to lysosomes and rapidly degrade internalized Hb. Whereas Hb transport to lysosomes and its degradation is significantly inhibited in cells overexpressing Rab7:T21N, a GDP locked mutant of Rab7. Moreover, cells overexpressing Rab7:T21N grow at a slower rate (<50%) compared with control Leishmania. Addition of exogenous hemin recovers the growth of Rab7:T21N mutant cells almost to the control level, suggesting that intracellular heme generated by Rab7-mediated Hb degradation is required for optimal growth of the parasites. Thus, our results identify a potential target which might be exploited to suppress the growth of Leishmania.

Mycobacterium tuberculosis secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex inhibit lipopolysaccharide-induced NF-kappaB transactivation by downregulation of reactive oxidative species (ROS) production.

Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people annually and is considered one of the most successful intracellular pathogens to persist inside the host macrophage. Recent studies have implicated the role of RD-1 region of Mtb genome in the mycobacterial pathogenesis. The role of RD-1-encoded secretory proteins of Mtb in modulation of macrophage function has not been investigated in detail. Here we show that RD-1 encoded two major secretory proteins, namely, culture filtrate protein-10 kDa (CFP-10) and early secreted antigenic target-6 kDa (ESAT-6), and their 1:1 CFP-10:ESAT6 complex inhibit production of reactive oxidative species (ROS) in RAW264.7 cells. These proteins also downregulated the bacterial lipopolysaccharide (LPS)-induced ROS production, which, in turn, downregulated LPS-induced nuclear factor-kappaB (NF-kappaB) p65 DNA-binding activity, as well as inhibited the NF-kappaB-dependent reporter gene (chloramphenicol acetyl transferase) expression in the treated macrophages. Moreover, addition of N-acetyl cysteine, which is a scavenger of ROS, also inhibited LPS-induced reporter gene expression by scavenging the ROS, thereby preventing NF-kappaB transactivation. These studies indicate that the secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex of Mtb can inhibit LPS-induced NF-kappaB-dependent gene expression via downregulation of ROS production.

Mycobacterium tuberculosis 6-kDa early secreted antigenic target (ESAT-6) protein downregulates lipopolysaccharide induced c-myc expression by modulating the extracellular signal regulated kinases 1/2.

BACKGROUND: Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people every year. The persistence of the pathogenic mycobacteria inside the macrophage occurs through modulation of host cell signaling which allows them, unlike the other non-pathogenic species, to survive inside the host. The secretory proteins of M. tuberculosis have gained attention in recent years both as vaccine candidates and diagnostic tools; they target the immune system and trigger a putatively protective response; however, they may also be involved in the clinical symptoms of the disease. RESULTS: Our studies showed that RD-1-encoded secretory protein ESAT-6 is involved in modulation of the mitogen-activated protein (MAP) kinase-signaling pathway inside the macrophage. ESAT-6 induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the cytoplasm but not in the nucleus, which normally is the case for MAP kinases. ESAT-6 also antagonized LPS-induced ERK1/2 phosphorylation in the nucleus. Stimulation of cells by ESAT-6 along with sodium orthovanadate (a tyrosine phosphatase inhibitor) restored phosphorylation of ERK1/2 in the nucleus, suggesting active dephosphorylation of ERK1/2 by some putative phosphatase(s) in the nucleus. Further, ESAT-6 was found to down regulate the expression of LPS-inducible gene c-myc in an ERK1/2-dependent manner. CONCLUSION: This study showed the effect of secretory proteins of M. tuberculosis in the modulation of macrophage signaling pathways particularly ERK1/2 MAP kinase pathway. This modulation appears to be achieved by limiting the ERK1/2 activation in the nucleus which ultimately affects the macrophage gene expression. This could be a mechanism by which secretory proteins of Mtb might modulate gene expression inside the macrophages.

IL-6 and IL-12 specifically regulate the expression of Rab5 and Rab7 via distinct signaling pathways.

Recent studies have shown that phagosome maturation depends on the balance between pro-inflammatory and anti-inflammatory cytokines, indicating that cytokine modulates phagosome maturation. However, the mechanism of cytokine-mediated modulation of intracellular trafficking remains to be elucidated. Here, we have shown that treatment of macrophages with IL-6 specifically induce the expression of Rab5 through the activation of extracellular signal-regulated kinase, whereas IL-12 exclusively upregulate the expression of Rab7 through the activation of p38 MAPK. We have cloned the 5'-flanking regions of the rab5c or rab7 into the promoterless reporter vector. Our results have shown that cells transfected with rab5c chimera are transactivated by IL-6, and IL-12 specifically transactivates cells containing rab7 chimera. Moreover, our results also show that IL-12 induces lysosomal transport, whereas IL-6 stimulates the fusion between early compartments in macrophages and accordingly modulates Salmonella trafficking and survival in macrophages. This is the first demonstration showing that cytokine differentially regulates endocytic trafficking by controlling the expression of appropriate Rab GTPase, and provides insight into the mechanism of cytokine-mediated regulation of intracellular trafficking.