IL6/sIL6R regulates TNFα-inflammatory response in synovial fibroblasts through modulation of transcriptional and post-transcriptional mechanisms.

INTRODUCTION: The clinical efficacy of specific interleukin-6 inhibitors has confirmed the central role of IL6 in rheumatoid arthritis (RA). However the local role of IL6, in particular in synovial fibroblasts (SF) as a direct cellular target to IL6/sIL6R signal is not well characterized. The purpose of the study was to characterize the crosstalk between TNFα and IL6/sIL6R signaling to the effector pro-inflammatory response of SF. METHODS: SF lines were stimulated with either TNFα, IL6/sIL6R, or both together, for the time and dose indicated for each experiment, and where indicated, cells were treated with inhibitors actinomycin D, adalimumab, ruxolitinib and cycloheximide. mRNA expression of cytokines, chemokines and matrix metalloproteases (MMPs) were analyzed by quantitative RT-PCR. Level of IL8/CXCL8 and CCL8 in culture supernatants was measured by ELISA. Mononuclear and polymorphonuclear cells migration assays were assessed by transwell using conditioned medium from SF cultures. Statistical analyses were performed as indicated in the corresponding figure legends and a p-value < 0.05 was considered statistically significant. RESULTS: The stimulation of SF with IL6/sIL6R and TNFα, cooperatively promotes the expression of mono- and lymphocytic chemokines such as IL6, CCL8 and CCL2, as well as matrix degrading enzymes such as MMP1, while inhibiting the induction of central neutrophil chemokines such as IL8/CXCL8. These changes in the pattern of chemokines expression resulted in reduced polymorphonuclear (PMN) and increased mononuclear cells (MNC) chemoattraction by SF. Mechanistic analyses of the temporal expression of genes demonstrated that the cooperative regulation mediated by these two factors is mostly induced through de novo transcriptional mechanisms activated by IL6/sIL6R. Furthermore, we also demonstrate that TNFα and IL6/sIL6R cooperation is partially mediated by the expression of secondary factors signaling through JAK/STAT pathways. CONCLUSIONS: These results point out to a highly orchestrated response to IL6 in TNFα-induced SF and provide additional insights into the role of IL6/sIL6R in the context of RA, highlighting the contribution of IL6/sIL6R to the interplay of SF with other inflammatory cells.

Senescent synovial fibroblasts accumulate prematurely in rheumatoid arthritis tissues and display an enhanced inflammatory phenotype.

BACKGROUND: Accumulation of senescent cells has been associated with pro-inflammatory effects with deleterious consequences in different human diseases. The purpose of this study was to analyze cell senescence in human synovial tissues (ST), and its impact on the pro-inflammatory function of synovial fibroblasts (SF). RESULTS: The expression of the senescence marker p16INK4a (p16) was analyzed by immunohistochemistry in rheumatoid arthritis (RA), osteoarthritis (OA), and normal ST from variably aged donors. The proportion of p16(+) senescent cells in normal ST from older donors was higher than from younger ones. Although older RA and OA ST showed proportions of senescent cells similar to older normal ST, senescence was increased in younger RA ST compared to age-matched normal ST. The percentage of senescent SA-ß-gal(+) SF after 14 days in culture positively correlated with donor's age. Initial exposure to H2O2 or TNFα enhanced SF senescence and increased mRNA expression of IL6, CXCL8, CCL2 and MMP3 and proteins secretion. Senescent SF show a heightened IL6, CXCL8 and MMP3 mRNA and IL-6 and IL-8 protein expression response upon further challenge with TNFα. Treatment of senescent SF with the senolytic drug fenofibrate normalized IL6, CXCL8 and CCL2 mRNA expression. CONCLUSIONS: Accumulation of senescent cells in ST increases in normal aging and prematurely in RA patients. Senescence of cultured SF is accelerated upon exposure to TNFα or oxidative stress and may contribute to the pathogenesis of synovitis by increasing the production of pro-inflammatory mediators.

Hif-1α Knockdown Reduces Glycolytic Metabolism and Induces Cell Death of Human Synovial Fibroblasts Under Normoxic Conditions.

Increased glycolysis and HIF-1α activity are characteristics of cells under hypoxic or inflammatory conditions. Besides, in normal O2 environments, elevated rates of glycolysis support critical cellular mechanisms such as cell survival. The purpose of this study was to analyze the contribution of HIF-1α to the energy metabolism and survival of human synovial fibroblasts (SF) under normoxic conditions. HIF-1α was silenced using lentiviral vectors or small-interfering RNA (siRNA) duplexes. Expression analysis by qRT-PCR and western blot of known HIF-1α target genes in hypoxia demonstrated the presence of functional HIF-1α in normoxic SF and confirmed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a HIF-1α target even in normoxia. HIF-1α silencing induced apoptotic cell death in cultured SF and, similarly, treatment with glycolytic, but not with OXPHOS inhibitors, induced SF death. Finally, in vivo HIF-1α targeting by siRNA showed a significant reduction in the viability of human SF engrafted into a murine air pouch. Our results demonstrate that SF are highly dependent on glycolytic metabolism and that HIF-1α plays a regulatory role in glycolysis even under aerobic conditions. Local targeting of HIF-1α provides a feasible strategy to reduce SF hyperplasia in chronic arthritic diseases.

The Role of the Transcriptional Regulation of Stromal Cells in Chronic Inflammation.

Chronic inflammation is a common process connecting pathologies that vary in their etiology and pathogenesis such as cancer, autoimmune diseases, and infections. The response of the immune system to tissue damage involves a carefully choreographed series of cellular interactions between immune and non-immune cells. In recent years, it has become clear that stromal resident cells have an essential role perpetuating the inflammatory environment and dictating in many cases the outcome of inflammatory based pathologies. Signal transduction pathways remain the main focus of study to understand how stimuli contribute to perpetuating the inflammatory response, mainly due to their potential role as therapeutic targets. However, molecular events orchestrated in the nucleus by transcription factors add additional levels of complexity and may be equally important for understanding the phenotypic differences of activated stromal components during the chronic inflammatory process. In this review, we focus on the contribution of transcription factors to the selective regulation of inducible proinflammatory genes, with special attention given to the regulation of the stromal fibroblastic cell function and response.

Enforcing the pause: transcription factor Sp3 limits productive elongation by RNA polymerase II.

The transition of paused RNA polymerase II into productive elongation is a highly dynamic process that serves to fine-tune gene expression in response to changing cellular environments. We have recently reported that the transcription factor Sp3 inhibits the transition of paused RNA Pol II to productive elongation at the promoter of the cyclin-dependent kinase inhibitor p21(CIP1) and other Sp3-repressed genes. Our studies support the view that Sp3 has three modes of action: activation, SUMO-Sp3-mediated heterochromatin silencing and SUMO-independent inhibition of elongation. At the p21(CIP1) promoter, binding of the positive elongation factor P-TEFb kinase was not affected by Sp3. In contrast, Sp3 promoted binding of the protein phosphatase PP1 to the p21(CIP1) promoter, suggesting that Sp3-dependent regulation of the local balance between kinase and phosphatase activities may contribute to gene expression. Our findings show that the transition of paused RNA Pol II to productive elongation is an important step regulated by both promoter-specific activators and repressors to finely modulate mRNA expression levels.

Transcription factor Sp3 represses expression of p21CIP¹ via inhibition of productive elongation by RNA polymerase II.

Like that of many protein-coding genes, expression of the p21(CIP1) cell cycle inhibitor is regulated at the level of transcription elongation. While many transcriptional activators have been shown to stimulate elongation, the mechanisms by which promoter-specific repressors regulate pausing and elongation by RNA polymerase II (RNA PolII) are not well described. Here we report that the transcription factor Sp3 inhibits basal p21(CIP1) gene expression by promoter-bound RNA PolII. Knockdown of Sp3 led to increased p21(CIP1) mRNA levels and reduced occupancy of the negative elongation factor (NELF) at the p21(CIP1) promoter, although the level of binding of the positive transcription elongation factor b (P-TEFb) kinase was not increased. Sp3 depletion correlated with increased H3K36me3 and H2Bub1, two histone modifications associated with transcription elongation. Further, Sp3 was shown to promote the binding of protein phosphatase 1 (PP1) to the p21(CIP1) promoter, leading to reduced H3S10 phosphorylation, a finding consistent with Sp3-dependent regulation of the local balance between kinase and phosphatase activities. Analysis of other targets of Sp3-mediated repression suggests that, in addition to previously described SUMO modification-dependent chromatin-silencing mechanisms, inhibition of the transition of paused RNA PolII to productive elongation, described here for p21(CIP1), is a general mechanism by which transcription factor Sp3 fine-tunes gene expression.

Sp4-dependent repression of neurotrophin-3 limits dendritic branching.

Regulation of neuronal gene expression is critical to establish functional connections in the mammalian nervous system. The transcription factor Sp4 regulates dendritic patterning during cerebellar granule neuron development by limiting branching and promoting activity-dependent pruning. Here, we investigate neurotrophin-3 (NT3) as a target gene important for Sp4-dependent dendritic morphogenesis. We found that Sp4 overexpression reduced NT3 promoter activity whereas knockdown of Sp4 increased NT3 promoter activity and mRNA. Moreover, Sp4 bound to the NT3 promoter in vivo, supporting a direct role for Sp4 as a repressor of NT3 expression. Addition of exogenous NT3 promoted dendritic branching in cerebellar granule neurons. Furthermore, sequestering NT3 blocked the continued addition of dendritic branches observed upon Sp4 knockdown, but had no effect on dendrite pruning. These findings demonstrate that, during cerebellar granule neuron development, Sp4-dependent repression of neurotrophin-3 is required to limit dendritic branching and thereby promote acquisition of the mature dendritic pattern.

Sp1 and Sp3 regulate transcription of the cyclin-dependent kinase 5 regulatory subunit 2 (p39) promoter in neuronal cells.

Cyclin-dependent kinase 5 (cdk5) activity is critical for development and function of the nervous system. Cdk5 activity is dependent on association with the regulators p35 and p39 whose expression is highly regulated in the developing nervous system.We have identified a small 200 bp fragment of the p39 promoter that is sufficient for cell type-specific expression in neuronal cells. Mutational analysis revealed that a cluster of predicted binding sites for Sp1, AP-1/CREB/ATF and E box-binding transcription factors is essential for full activity of the p39 promoter. Electrophoretic mobility shift assays revealed that Sp1 and Sp3 bound to sequences required for p39 promoter function and chromatin immunoprecipitation assays confirmed binding of these proteins to the endogenous p39 promoter. Furthermore, depletion of either Sp1 or Sp3 by siRNA reduced expression from the p39 promoter. Our data suggest that the ubiquitously expressed transcription factors Sp1 and Sp3 regulate transcription of the cdk5 regulator p39 in neuronal cells, possibly in cooperation with tissue-specific transcription factors.

Direct binding of CoREST1 to SUMO-2/3 contributes to gene-specific repression by the LSD1/CoREST1/HDAC complex.

Posttranslational modification of transcription factors by the small ubiquitin-related modifier SUMO is associated with transcriptional repression, but the underlying mechanisms remain incompletely described. We have identified binding of the LSD1/CoREST1/HDAC corepressor complex to SUMO-2. Here we show that CoREST1 binds directly and noncovalently to SUMO-2, but not SUMO-1, and CoREST1 bridges binding of the histone demethylase LSD1 to SUMO-2. Depletion of SUMO-2/3 conjugates led to transcriptional derepression, reduced occupancy of CoREST1 and LSD1, and changes in histone methylation and acetylation at some, but not all, LSD1/CoREST1/HDAC target genes. We have identified a nonconsensus SUMO-interaction motif (SIM) in CoREST1 required for SUMO-2 binding, and we show that mutation of the CoREST1 SIM disrupted SUMO-2 binding and transcriptional repression of some neuronal-specific genes in nonneuronal cells. Our results reveal that direct interactions between CoREST1 and SUMO-2 mediate SUMO-dependent changes in chromatin structure and transcription that are important for cell-type-specific gene expression.

Regulation of transcription factor activity by SUMO modification.

Post-translational modification by SUMO is an important mechanism to regulate transcription. Sumoyla-tion has diverse effects on substrate activity, but in most cases reported to date sumoylation of transcription factors correlated with transcriptional repression. Here we describe general strategies to address how post-translational modification by SUMO regulates the activity of a DNA-binding transcription factor.