Radiation-Induced Senescence Reprograms Secretory and Metabolic Pathways in Colon Cancer HCT-116 Cells.

Understanding the global metabolic changes during the senescence of tumor cells can have implications for developing effective anti-cancer treatment strategies. Ionizing radiation (IR) was used to induce senescence in a human colon cancer cell line HCT-116 to examine secretome and metabolome profiles. Control proliferating and senescent cancer cells (SCC) exhibited distinct morphological differences and expression of senescent markers. Enhanced secretion of pro-inflammatory chemokines and IL-1, anti-inflammatory IL-27, and TGF-ß1 was observed in SCC. Significantly reduced levels of VEGF-A indicated anti-angiogenic activities of SCC. Elevated levels of tissue inhibitors of matrix metalloproteinases from SCC support the maintenance of the extracellular matrix. Adenylate and guanylate energy charge levels and redox components NAD and NADP and glutathione were maintained at near optimal levels indicating the viability of SCC. Significant accumulation of pyruvate, lactate, and suppression of the TCA cycle in SCC indicated aerobic glycolysis as the predominant energy source for SCC. Levels of several key amino acids decreased significantly, suggesting augmented utilization for protein synthesis and for use as intermediates for energy metabolism in SCC. These observations may provide a better understanding of cellular senescence basic mechanisms in tumor tissues and provide opportunities to improve cancer treatment.

Proinflammatory cytokine interferon-γ increases the expression of BANCR, a long non-coding RNA, in retinal pigment epithelial cells.

The inflammatory response may contribute to retinal pigment epithelial (RPE) dysfunction associated with the pathogenesis of age-related macular degeneration (AMD). We investigated whether the inflammatory response affects the expression of long coding RNAs (lncRNAs) in human RPE-derived ARPE-19 cells. This class of regulatory RNA molecules recently came to prominence due to their involvement in many pathophysiological processes. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1ß and TNF-α altered the expression several lncRNAs including BANCR in these cells. The cytokine responsible for increasing BANCR expression in ARPE-19 cells was found to be IFN-γ. BANCR expression induced by IFN-γ was suppressed when STAT1 phosphorylation was blocked by JAK inhibitor 1. Thus, proinflammatory cytokines could modulate the expression of lncRNAs in RPE cells and IFN-γ could upregulate the expression of BANCR by activating JAK-STAT1 signaling pathway.

Proinflammatory cytokines decrease the expression of genes critical for RPE function.

PURPOSE: Proinflammatory cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1ß) secreted by infiltrating lymphocytes or macrophages may play a role in triggering RPE dysfunction associated with age-related macular degeneration (AMD). Binding of these proinflammatory cytokines to their specific receptors residing on the RPE cell surface can activate signaling pathways that, in turn, may dysregulate cellular gene expression. The purpose of the present study was to investigate whether IFN-γ, TNF-α, and IL-1ß have an adverse effect on the expression of genes essential for RPE function, employing the RPE cell line ARPE-19 as a model system. METHODS: ARPE-19 cells were cultured for 3-4 months until they exhibited epithelial morphology and expressed mRNAs for visual cycle genes. The differentiated cells were treated with IFN-γ, TNF-α, and/or IL-1ß, and gene expression was analyzed with real-time PCR analysis. Western immunoblotting was employed for the detection of proteins. RESULTS: Proinflammatory cytokines (IFN-γ + TNF-α + IL-1ß) greatly increased the expression of chemokines and cytokines in cultured ARPE-19 cells that exhibited RPE characteristics. However, this response was accompanied by markedly decreased expression of genes important for RPE function, such as CDH1, RPE65, RDH5, RDH10, TYR, and MERTK. This was associated with decreased expression of the genes MITF, TRPM1, and TRPM3, as well as microRNAs miR-204 and miR-211, which are known to regulate RPE-specific gene expression. The decreased expression of the epithelial marker gene CDH1 was associated with increased expression of mesenchymal marker genes (CDH2, VIM, and CCND1) and epithelial-mesenchymal transition (EMT) promoting transcription factor genes (ZEB1 and SNAI1). CONCLUSIONS: RPE cells exposed to proinflammatory cytokines IFN-γ, TNF-α, and IL-1ß showed decreased expression of key genes involved in the visual cycle, epithelial morphology, and phagocytosis. This adverse effect of proinflammatory cytokines, which could be secreted by infiltrating lymphocytes or macrophages, on the expression of genes indispensable for RPE function may contribute to the RPE dysfunction implicated in AMD pathology.

Inflammatory cytokines regulate secretion of VEGF and chemokines by human conjunctival fibroblasts: Role in dysfunctional tear syndrome.

Ocular surface inflammation is one of the primary mechanisms associated with dysfunctional tear syndrome (DTS), also known as dry eye disease. DTS, more prevalent in older populations, causes ocular discomfort and visual disturbance due to dryness on the surface layer in the eye. We used human conjunctival fibroblast cultures (HCJVF) to investigate the effects of inflammatory cytokines IFN-γ, TNF-α and IL-1ß (ITI) on the secretions of VEGF and chemokines. Our results demonstrate the elevated secretion of angiogenic VEGF molecules by ITI without affecting anti-angiogenic molecules, PEDF, endostatin, thrombospondin and sVEGF-R1. The secretion of interferon-γ inducible chemokines, CXCL9, -10, -11 by HCJVF were significantly enhanced by ITI. Our in vitro study supports previously reported observations of elevated VEGF and chemokines in tear fluids of DTS patients, reiterating the role of inflammatory reactions in DTS.

Inflammatory Cytokines Induce Expression of Chemokines by Human Retinal Cells: Role in Chemokine Receptor Mediated Age-related Macular Degeneration.

Chemokine reeptor-3 (CCR-3) was shown to be associated with choroidal neovascularization (CNV) in age-related macular degeneration (AMD). AMD is a vision threatening retinal disease that affects the aging population world-wide. Retinal pigment epithelium and choroid in the posterior part of the retina are the key tissues targeted in the pathogenesis of CNV in AMD. We used human retinal pigment epithelial (HRPE) and choroidal fibroblast (HCHF) cells, prepared from aged adult human donor eyes, to evaluate the expression of major CCR-3 ligands, CCL-5, CCL -7, CCL-11,CCL-24 and CCL-26. Microarray analysis of gene expression in HRPE cells treated with inflammatory cytokine mix (ICM= IFN-γ+TNF-α+IL-1ß) revealed 75 and 23-fold increase in CCL-5 and CCL-7 respectively, but not CCL-11, CCL-24 and CCL-26. Chemokine secretion studies of the production of CCL5 and CCL7 by HRPE corroborated with the gene expression analysis data. When the HRPE cells were treated with either individual cytokines or the ICM, both CCL-5 and CCL-7 were produced in a dose dependent manner. Similar to the gene expression data, the ICM did not enhance HRPE production of CCL-11, CCL-24 and CCL-26. CCL-11 and CCL-26 were increased with IL-4 treatment and this HRPE production was augmented in the presence of TNF-α and IL1ß. When HCHF cells were treated with either individual cytokines or the ICM, both CCL-5 and CCL-7 were produced in a dose dependent fashion. IL-4 induced low levels of CCL-11 and CCL-26 in HCHF and this production was significantly enhanced by TNF-α. Under these conditions, neither HRPE nor HCHF were demonstrated to produce CCL-24. These data demonstrate that chronic inflammation triggers CCL-5 and CCL-7 release by HRPE and HCHF and the subsequent interactions with CCR3 may participate in pathologic processes in AMD.

Resveratrol attenuates CXCL11 expression induced by proinflammatory cytokines in retinal pigment epithelial cells.

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). RPE cells adjacent to drusen deposits in the AMD eye are known to contain CXCL11, a chemokine involved in inflammatory cell recruitment. We investigated the CXCL11 production by the human RPE (ARPE-19) cells under inflammatory conditions and tested its response to resveratrol, a naturally occurring anti-inflammatory antioxidant. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1ß and TNF-α highly increased CXCL11 mRNA expression and CXCL11 protein secretion by ARPE-19 cells. Resveratrol substantially inhibited the proinflammatory cytokines-induced CXCL11 production while partially blocking nuclear factor-κB activation. This inhibitory action of resveratrol was also observed for the cytokines-induced expression of chemokines CXCL9, CCL2 and CCL5. Our results indicate that resveratrol could potentially attenuate RPE inflammatory response implicated in the pathogenesis of AMD.

Ceramide inhibits connective tissue growth factor expression by human retinal pigment epithelial cells.

Connective tissue growth factor (CTGF) is known to be involved in retinal fibrotic disorders. We used human retinal pigment epithelial cells (HRPE), which play critical roles in retinal fibrosis, to examine the expression of CTGF and its regulation by ceramide and TGF-ß. Real-time PCR analysis showed downregulation of CTGF mRNA by C2 ceramide and upregulation by TGF-ß. C2 ceramide also inhibited constitutive and TGF-ß-enhanced CTGF secretion by HRPE cells. Predominant secretion (>80% of total) of CTGF from the apical side was observed in highly polarized HRPE cells. Fumonosin, an inhibitor of ceramide synthesis, stimulated CTGF secretion while 4HPR, an activator of ceramide synthesis, downregulated CTGF secretion. Based on these results demonstrating ceramide regulation of CTGF secretion by HRPE, we suggest that ceramide may have therapeutic potential for the treatment of retinal fibrotic diseases by inhibiting CTGF production.

Resveratrol Suppresses Expression of VEGF by Human Retinal Pigment Epithelial Cells: Potential Nutraceutical for Age-related Macular Degeneration.

Age-related macular degeneration (AMD) is a sight threating retinal eye disease that affects millions of aging individuals world-wide. Choroid-retinal pigment epithelium (RPE)-neuroretina axis in the posterior compartment of the eye is the primary site of AMD pathology. There are compelling evidence to indicate association of vascular endothelial growth factors (VEGF) to AMD. Here, we report the inhibitory actions of resveratrol (RSV) on inflammatory cytokine, TGF-ß and hypoxia induced VEGF secretion by human retinal pigment epithelial cells (HRPE). HRPE cultures prepared from aged human donor eyes were used for the studies in this report. HRPE secreted both VEGF-A and VEGF-C in small quantities constitutively. Stimulation with a mixture of inflammatory cytokines (IFN-γ, TNF-α, IL-1ß), significantly increased the secretion of both VEGF-A and VEGF-C. RSV, in a dose dependent (10-50 uM) manner, suppressed VEGF-A and VEGF-C secretion induced by inflammatory cytokines significantly. RT-PCR analysis indicated that effects of RSV on VEGF secretion were possibly due to decreased mRNA levels. TGF-ß and cobalt chloride (hypoxia mimic) also upregulated HRPE cell production of VEGF-A, and this was inhibited by RSV. In contrast, RSV had no effect on anti-angiogenic molecules, endostatin and pigment epithelial derived factor secretion. Studies using an in vitro scratch assay revealed that wound closure was also inhibited by RSV. These results demonstrate that RSV can suppress VEGF secretion induced by inflammatory cytokines, TGF-ß and hypoxia. Under pathological conditions, over expression of VEGF is known to worsen AMD. Therefore, RSV may be useful as nutraceutical in controlling pathological choroidal neovascularization processes in AMD.

Transforming growth factor-ß3 (TGF-ß3) knock-in ameliorates inflammation due to TGF-ß1 deficiency while promoting glucose tolerance.

Three homologues of TGF-ß exist in mammals as follows: TGF-ß1, TGF-ß2, and TGF-ß3. All three proteins share high homology in their amino acid sequence, yet each TGF-ß isoform has unique heterologous motifs that are highly conserved during evolution. Although these TGF-ß proteins share similar properties in vitro, isoform-specific properties have been suggested through in vivo studies and by the unique phenotypes for each TGF-ß knock-out mouse. To test our hypothesis that each of these homologues has nonredundant functions, and to identify such isoform-specific roles, we genetically exchanged the coding sequence of the mature TGF-ß1 ligand with a sequence from TGF-ß3 using targeted recombination to create chimeric TGF-ß1/3 knock-in mice (TGF-ß1(Lß3/Lß3)). In the TGF-ß1(Lß3/Lß3) mouse, localization and activation still occur through the TGF-ß1 latent associated peptide, but cell signaling is triggered through the TGF-ß3 ligand that binds to TGF-ß receptors. Unlike TGF-ß1(-/-) mice, the TGF-ß1(Lß3/Lß3) mice show neither embryonic lethality nor signs of multifocal inflammation, demonstrating that knock-in of the TGF-ß3 ligand can prevent the vasculogenesis defects and autoimmunity associated with TGF-ß1 deficiency. However, the TGF-ß1(Lß3/Lß3) mice have a shortened life span and display tooth and bone defects, indicating that the TGF-ß homologues are not completely interchangeable. Remarkably, the TGF-ß1(Lß3/Lß3) mice display an improved metabolic phenotype with reduced body weight gain and enhanced glucose tolerance by induction of beneficial changes to the white adipose tissue compartment. These findings reveal both redundant and unique nonoverlapping functional diversity in TGF-ß isoform signaling that has relevance to the design of therapeutics aimed at targeting the TGF-ß pathway in human disease.

Differential regulation of microRNA-146a and microRNA-146b-5p in human retinal pigment epithelial cells by interleukin-1ß, tumor necrosis factor-α, and interferon-γ.

PURPOSE: The inflammatory response of the retinal pigment epithelium (RPE) is implicated in the pathogenesis of age-related macular degeneration. The microRNAs miR-146a and miR-146b-5p can regulate the inflammatory process by attenuating cytokine signaling via the nuclear factor-κB pathway. The aim of the present study is to investigate the expression of miR-146a and miR-146b-5p in human RPE cells and their response to proinflammatory cytokines. METHODS: Confluent cultures of RPE cells established from adult human donor eyes were treated with the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß. The expression of microRNAs was analyzed by real-time PCR using total RNA fraction. The retinal pigment epithelial cell line ARPE-19 was employed to analyze the promoter activity of the genes encoding miR-146a and miR-146b-5p. STAT1-binding activity of oligonucleotides was analyzed by electrophoretic mobility shift assay. ARPE-19 cells were transiently transfected with miR-146a and miR-146b-5p mimics for the analysis of IRAK1 expression by western immunoblotting. RESULTS: Real-time PCR analysis showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to proinflammatory cytokines (IFN-γ + TNF-α + IL-1ß) by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10, and IL-6, and a decrease in that for HMOX1. The miR-146a induction was more dependent on IL-1ß, since its omission from the cytokine mix resulted in a greatly reduced response. Similarly, the induction of miR-146b-5p was more dependent on IFN-γ, since its omission from the cytokine mix minimized the effect. In addition, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of the JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with either miR-146a mimic or miR-146b-5p mimic. CONCLUSIONS: Our results clearly show that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is highly induced by proinflammatory cytokines (IFN-γ + TNF-α + IL-1ß). The induction of miR-146a showed a dependency on IL-1ß, while that of miR-146b-5p on IFN-γ. Our results show for the first time that miR-146b-5p expression is regulated by IFN-γ, potentially via the JAK/STAT pathway. These two microRNAs could play a role in inflammatory processes underlying age-related macular degeneration or other retinal degenerative diseases through their ability to negatively regulate the nuclear factor-κB pathway by targeting the expression of IRAK1.

The tellurium redox immunomodulating compound AS101 inhibits IL-1ß-activated inflammation in the human retinal pigment epithelium.

PURPOSE: AS101 is a non-toxic organotellurium-IV compound with demonstrated immunomodulating activity in vitro and in vivo. Inflammatory responses are attributed to the pathophysiology of numerous ocular diseases. In this study, we wished to elucidate whether AS101 could mitigate pro-inflammatory activity in human retinal pigment epithelial (RPE) cells, which are heavily involved in ocular immune responses, induced by pro-inflammatory IL-ß activity. METHODS: Primary and transformed RPE cells treated with varying concentrations of AS101 were used in this study. Real-time PCR and ELISA assays were used to detect cytokine/chemokine mRNA expression and protein production. Western blot was used to detect changes in the NFκB pathway. Cell viability and proliferation were detected using a Vi-Cell XR cell counter. To measure the cytoprotective capacity of AS101, cell numbers were compared between cells treated with IL-1ß or lipopolysaccharide (LPS) and cells treated with IL-1ß or LPS in the presence of AS101. RESULTS: AS101 inhibited IL-1ß-induced mRNA expression and protein production of IL-6 and IL-8 in RPE cells. The viability of RPE cells treated with IL-1ß and LPS was unaffected. AS101 slightly inhibited RPE cell growth in the presence of higher levels of IL-1ß. Also, AS101 downregulated the IL-1ß activity by inhibiting the phosphorylation of p65, an NFκB subunit. CONCLUSIONS: The results demonstrate that AS101 reduces IL-1ß-induced inflammatory responses in the RPE. In previous studies, AS101 exhibited therapeutic effects in various disease models and was a safe profile in clinical trials. These results suggest that AS101 may have potent anti-inflammatory potential in the eye and confer the downregulation of RPE inflammatory responses in a pathological environment.

Transforming growth factor-ß regulates the expression of anosmin (KAL-1) in human retinal pigment epithelial cells.

In a microarray analysis of human retinal pigment epithelial cells (HRPE) treated with TGF-ß, in addition to the alteration of a number of known Extracellular matrix (ECM)-related genes regulated by TGF-ß, we found a significant increase in the expression of Kallmann Syndrome (KAL)-1 gene, that codes for the protein anosmin-1. Enhanced expression of KAL-1 by TGF-ß was validated by real-time PCR analysis. In in vitro experiments, TGF-ß receptor inhibitor abolished TGF-ß-induced expression of KAL-1. Immunofluorescence staining showed increased presence of anosmin-1 in TGF-ß treated HRPE cells, with distinct localization at the intercellular junctions. Treatment of HRPE cells with TGF-ß enhanced secretion of anosmin-1 and the release of anosmin-1 was further augmented by heparin sulfate. Enhanced secretion of anosmin-1 in the presence of TGF-ß and heparin was also observed in other ocular cells such as corneal epithelial and corneal fibroblast cultures. The role of anosmin-1, a protein with adhesion functions, in retinal structure, function and pathology has not been known and remains to be investigated.

Regulation of VEGF expression in human retinal cells by cytokines: implications for the role of inflammation in age-related macular degeneration.

Chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). Choroidal neovascularization (CNV) observed in exudative form of AMD results in vision loss. Human retinal pigment epithelial cell (HRPE) layer and choroidal tissue are the primary pathological sites in AMD. Pathological and therapeutic evidences have strongly indicated the vascular endothelial growth factor (VEGF) molecules as critical components in CNV pathogenesis. In these studies, we used human primary HRPE and choroidal fibroblast cells (HCHF) prepared from adult donor eyes. The effects of inflammatory cytokine (IFN-γ+ TNF-α+IL-1ß) mix (ICM) on global gene expression profiles in HRPE cells, revealed 10- and 9-fold increase in VEGF-A and VEGF-C expression, respectively. The microarray results were validated by quantitative RT-PCR and secretion of VEGFs proteins. IL-1ß is the most potent in inducing VEGFs secretion followed by IFN-γ and TNF-α, and the secretion was more effective in the presence of 2 and 3 cytokines. NF-κB and JAK-STAT pathway, but not HIF-1α, Sp-1, Sp-3, and STAT-3, transcription factors were upregulated and translocated to nucleus by ICM treatment. The mRNA levels of VEGF-A and VEGF-C and secretion of these proteins were also significantly enhanced by ICM in HCHF cells. The secretion of other angiogenic molecules, PEDF, SDF-1α, endostatin, and angiopoietins was not affected by ICM. Our results show that the inflammatory cytokines enhance secretion of VEGF-A and VEGF-C by HRPE and HCHF cells. These studies indicate that VEGFs secreted by these cells initiate and promote pathological choroidal and retinal noevascularization processes in AMD.

Inflammatory cytokines regulate microRNA-155 expression in human retinal pigment epithelial cells by activating JAK/STAT pathway.

Inflammatory response of the retinal pigment epithelium plays a critical role in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. Our previous studies have shown that human retinal pigment epithelial (HRPE) cells, established from adult donor eyes, respond to inflammatory cytokines by enhancing the expression of a number of cytokines and chemokines. To investigate the role of microRNA (miRNA) in regulating this response, we performed microarray analysis of miRNA expression in HRPE cells exposed to inflammatory cytokine mix (IFN-γ+TNF-α+IL-1ß). Microarray analysis revealed ∼11-fold increase in miR-155 expression, which was validated by real-time PCR analysis. The miR-155 expression was enhanced when the cells were treated individually with IFN-γ, TNF-α or IL-1ß, but combinations of the cytokines exaggerated the effect. The increase in miR-155 expression by the inflammatory cytokines was associated with an increase in STAT1 activation as well as an increase in protein binding to putative STAT1 binding elements present in the MIR155 gene promoter region. All these activities were effectively blocked by JAK inhibitor 1. Our results show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT signaling pathway.

MicroRNA expression in human retinal pigment epithelial (ARPE-19) cells: increased expression of microRNA-9 by N-(4-hydroxyphenyl)retinamide.

PURPOSE: MicroRNAs (miRNAs) are important regulators of many cellular functions due to their ability to target mRNAs for degradation or translational inhibition. Previous studies have reported that the expression of microRNA-9 (miR-9) is regulated by retinoic acid and reactive oxygen species (ROS). We have previously shown that N-(4-hydroxyphenyl)-retinamide (4HPR), a retinoic acid derivative, induces ROS generation and apoptosis in cultured human retinal pigment epithelial (RPE) cells, known as ARPE-19 cells. The aim of the present study was to investigate the expression of miR-9 in ARPE-19 cells in response to 4HPR treatment, and to identify other miRNAs normally expressed in these cells. METHODS: ARPE-19 cells in culture were treated with 4HPR, the total RNA fractions were isolated, and the expression of various miRNAs and mRNAs was analyzed using real-time PCR. The miRNA expression profile of ARPE-19 cells was analyzed using microarray hybridization. RESULTS: Treatment of ARPE-19 cells with 4HPR resulted in apoptosis characterized by the increased expression of HMOX1 and GADD153 genes. A twofold increase in the expression of miR-9 was also observed during this response. Potential binding sites for the transcription factors encoded by CEBPA and CEBPB genes were found to be present in the putative promoter regions of all three genes encoding miR-9. 4HPR-induced miR-9 expression was associated with parallel increases in the expression of these transcription factor genes. 5-Aza-2'-deoxycytidine, a methyl transferase inhibitor, also increased the expression of miR-9 in ARPE-19 cells. Microarray hybridization analysis identified let-7b, let-7a, miR-125b, miR-24, miR-320, miR-23b, let-7e, and let-7d as the most abundant miRNAs normally expressed in ARPE-19 cells. These miRNAs are known to regulate cell growth, differentiation or development. The 4HPR treatment increased the expression of miR-16, miR-26b, miR-23a, and miR-15b in ARPE-19 cells, although these increases were modest when compared to the increase in the expression of miR-9. CONCLUSIONS: Our studies demonstrate that miR-9 is expressed in the RPE cell line ARPE-19, and its expression is increased by a retinoic acid derivative and by an inhibitor of promoter hypermethylation. Several miRNAs with inherent ability to regulate cell growth, differentiation and development are also normally expressed in ARPE-19 cells. Thus, miR-9 and other miRNAs could be important in maintaining RPE cell function.

Conditional overexpression of TGF-beta1 disrupts mouse salivary gland development and function.

Transforming growth factor-beta (TGF-beta) signaling is known to affect salivary gland physiology by influencing branching morphogenesis, regulating ECM deposition, and controlling immune homeostasis. To study the role of TGF-beta1 in the salivary gland, we created a transgenic mouse (beta1(glo)) that conditionally overexpresses active TGF-beta1 upon genomic recombination by Cre recombinase. beta1(glo) mice were bred with an MMTV (mouse mammary tumor virus)-Cre (MC) transgenic line that expresses the Cre recombinase predominantly in the secretory cells of both the mammary and salivary glands. Although most of the double positive (beta1(glo)/MC) pups die either in utero or just after birth, clear defects in salivary gland morphogenesis such as reduced branching and increased mesenchyme could be seen. Those beta1(glo)/MC mice that survived into adulthood, however, had hyposalivation due to salivary gland fibrosis and acinar atrophy. Increased TGF-beta signaling was observed in the salivary gland with elevated phosphorylation of Smad2 and concomitant increase in ECM deposition. In particular, aberrant TGF-beta1 overexpression caused salivary gland hypofunction in this mouse model because of the replacement of normal glandular parenchyma with interstitial fibrous tissue. These results further implicate TGF-beta in pathological cases of salivary gland inflammation and fibrosis that occur with chronic infections in the glands or with the autoimmune disease, Sjögren's syndrome, or with radiation therapy given to head-and-neck cancer patients.

IL-11 expression in retinal and corneal cells is regulated by interferon-gamma.

Interleukin-11 (IL-11) is an anti-apoptotic, anti-inflammatory cytokine with hematopoietic potential. The expression and protective actions of IL-11 have not been explored in the eye. The expression of IL-11 in primary cultures of human retinal pigment epithelial (HRPE) and human corneal fibroblast (HCRF) cells were evaluated in these studies. Constitutive secretion of IL-11 was not observed in either HRPE or HCRF. TNF-alpha+IL-1 induced IL-11 secretion and this production was inhibited by NFkappaB pathway inhibitors. IFN-gamma significantly inhibited TNF-alpha and IL-1 induced IL-11 secretion and inhibitors of JAK-STAT pathway reversed this inhibition. TGF-beta induced IL-11 secretion that was blocked by TGF-beta receptor 1 inhibitor but not by IFN-gamma. RT-PCR analysis confirmed the effects of IL-1, TNF-alpha, IFN-gamma and TGF-beta on IL-11 secretion at mRNA levels. Our results demonstrate that IL-11 is dramatically up regulated in retina and cornea cells and that IFN-gamma is a physiological inhibitor of IL-11 expression.

Anosmin-1 involved in neuronal cell migration is hypoxia inducible and cancer regulated.

Functional expression of KAL1 gene is critical in the migration of GnRH neurons from the olfactory placode to the hypothalamus in embryogenesis. This gene thus far has not been shown to play a functional role in any other physiological or pathological process either in the developed brain or in peripheral tissues. We show here that KAL1 gene expression is decreased in early stage and increased in later stages of cancers. Screening of colon, lung and ovarian cancer cDNA panels indicated significant decrease in KAL1 expression in comparison to corresponding uninvolved tissues. However, KAL1 expression increased with the progression of cancer from early (I and II) stages to later (III and IV) stages of the cancer. There was a direct correlation between the TGFbeta and KAL1 expression in colon cancer cDNA. Using colon cancer cell lines, we showed that TGFbeta induces KAL1 gene expression and secretion of anosmin-1 protein (KAL1 coded protein). We further report that hypoxia induces anosmin-1 expression; anosmin-1 protects cancer cells from apoptosis activated by hypoxia and increases cancer cell mobility. Using siRNA technique we found that KAL1 expression following hypoxia is hypoxia-inducible factor (HIF-1)alpha dependent. Our results suggest that KAL1 gene expression plays an important role in cancer metastasis and protection from apoptosis.

Functional identification of a novel transport system for endogenous and synthetic opioid peptides in the rabbit conjunctival epithelial cell line CJVE.

PURPOSE: To investigate whether conjunctival epithelial cells express transport processes for opioid peptides. METHODS: We monitored the uptake of [(3)H]deltorphin II and [(3)H]DADLE, two hydrolysis-resistant synthetic opioid peptides, in the rabbit conjunctival epithelial cell line CJVE and elucidated the characteristics of the uptake process. RESULTS: CJVE cells express robust uptake activity for deltorphin II and DADLE. Both opioid peptides compete with each other for transport. Several endogenous and synthetic opioid peptides, but not non-peptide opioid antagonists, are recognized by the transport process. Though various peptides inhibit the uptake of deltorphin II and DADLE in a similar manner, the uptake of deltorphin II is partly Na(+)-dependent whereas that of DADLE mostly Na(+)-independent. The transport process shows high affinity for many endogenous/synthetic opioid peptides. Functional features reveal that this transport process may be distinct from the opioid peptide transport system described in the retinal pigment epithelial cell line ARPE-19 and also from the organic anion transporting polypeptides, which are known to transport opioid peptides. CONCLUSIONS: CJVE cells express a novel, hitherto unknown transport process for endogenous/synthetic opioid peptides. This new transport process may offer an effective delivery route for opioid peptide drugs to the posterior segment of the eye.

IFN-gamma acts as anti-angiogenic cytokine in the human cornea by regulating the expression of VEGF-A and sVEGF-R1.

Inflammatory processes within the cornea are known to be associated with corneal neovascularization (CN). We examined the effects of inflammatory mediators on the expression of angiogenic factors by corneal cells. TNF-alpha and IL-1 induced VEGF-A secretion by corneal fibroblasts (HCRF) and this was inhibited significantly by IFN-gamma. Constitutively secreted VEGF-A by corneal epithelial cells (HCE) was not affected by these cytokines. Moreover, sVEGF-R1(sFlt-1) secretion by HCRF was stimulated significantly by IFN-gamma. JAK-STAT pathway inhibitor reversed the effects of IFN-gamma on VEGF-A and sFlt-1 secretion by HCRF. RT-PCR analysis showed that IFN-gamma influences the expression of VEGF-A and sFlt-1 by affecting their mRNA level. IFN-gamma inhibited TGF-beta induced VEGF-A secretion but not sVEGF-R1 secretion. This is the first report demonstrating the inhibitory and stimulatory effects of IFN-gamma on VEGF-A and sFlt-1 secretion, respectively. Our results suggest that IFN-gamma acts as an anti-angiogenic cytokine in the human cornea.