Inhibition of BATF/JUN transcriptional activity protects against osteoarthritic cartilage destruction

ObjectiveThe basic leucine zipper transcription factor, ATF-like (BATF), a member of the Activator protein-1 family, promotes transcriptional activation or repression, depending on the interacting partners (JUN-B or C-JUN). Here, we investigated whether the BATF/JUN complex exerts regulatory effects on catabolic and anabolic gene expression in chondrocytes and contributes to the pathogenesis of osteoarthritis (OA).MethodsPrimary cultured mouse chondrocytes were treated with proinflammatory cytokines (interleukin-1β, IL-6 or tumour necrosis factor-α) or infected with adenoviruses carrying the Batf gene (Ad-Batf). Expression of BATF and JUN was examined in human and mouse experimental OA cartilage samples. Experimental OA in mice was induced by destabilisation of the medial meniscus or intra-articular injection of Ad-Batf. The chromatin immunoprecipitation assay was used to examine the binding of BATF and JUN to the promoter regions of candidate genes.ResultsOverexpression of BATF, which forms a heterodimeric complex with JUN-B and C-JUN, induced upregulation of matrix-degrading enzymes and downregulation of cartilage matrix molecules in chondrocytes. BATF expression in mouse joint tissues promoted OA cartilage destruction, and conversely, knockout of Batf in mice suppressed experimental OA. Pharmacological inhibition of BATF/JUN transcriptional activity reduced the expression of matrix-degrading enzymes and protected against experimental OA in mice.ConclusionsBATF/JUN-B and BATF/C-JUN complexes play important roles in OA cartilage destruction through regulating anabolic and catabolic gene expression in chondrocytes. Our findings collectively support the utility of BATF as a therapeutic target for OA.

β2-Adrenergic receptors inhibit the expression of collagen type II in growth plate chondrocytes by stimulating the AP-1 factor Jun-B

The sympathetic nervous system can regulate both osteoblast and chondrocyte growth and activity through β2-adrenergic receptors (β2-AR). We have shown previously that β2-AR activate both adenylyl cyclase and mitogen-activated protein kinases ERK1/2 in growth plate chondrocytes prepared from ribs of embryonic E18.5 mice. Here we examined β2-AR inhibition of collagen type II (Col II) expression in growth plate chondrocytes and the molecular pathways involved. Stimulation of β2-AR by isoproterenol inhibited Col II mRNA and protein levels by ∼50% beginning at 2 h, with both remaining suppressed over 24 h. This inhibition was blocked by propranolol and inhibitors of either MEK1 or PKA. Isoproterenol stimulated an AP-1-luciferase reporter and increased the expression of AP-1 factors c-Fos, Fra-1, Fra-2, c-Jun, and Jun-B but had no effect on Jun-D. Stimulation of AP-1 activity was blocked by inhibitors of MEK1 or PKA. siRNA inhibition of AP-1 factors showed that depletion of only Jun-B attenuated isoproterenol-mediated inhibition of Col II. Transfection with jun-B or c-fos showed selective inhibition of Col II mRNA and a Col II luciferase reporter construct by jun-B. Isoproterenol as well as jun-B overexpression in the chondrocytes also inhibited the expression of Sox-6 mRNA and protein, and depletion of Jun-B abrogated β2-AR inhibition of Sox-6. Collectively, these findings demonstrate regulation of chondrocyte differentiation through β2-AR mediated by ERK1/2 and PKA stimulation of the AP-1 factor Jun-B that inhibits the expression of Sox-6 and Col II.

Hodgkin’s Lymphoma Reed Sternberg Cells over Express the T-Cell Inhibitory Carbohydrate-Binding Lectin, Galectin 1: Role of AP-1 and Likely Mechanism of Tumor Immune Escape.

Primary Hodgkin lymphomas (HL) contain small numbers of neoplastic Reed-Sternberg (RS) cells within an extensive host inflammatory infiltrate, which includes few cytotoxic T-cells. The lack of an effective host anti-tumor response prompts speculation regarding the mechanisms that HL RS cells use to evade immune surveillance. To identify novel HL-specific T-cell inhibitory molecules, we compared the gene expression profiles of a series of HL and diffuse large B-cell lymphoma (DLBCL) cell lines. The HL cell lines overexpressed galectin-1 (GAL1), a carbohydrate-binding lectin that selectively induces the apoptosis of cytotoxic T cells. GAL1 mRNA abundance was 5- to 33- fold higher in HL cell lines than in DLBCL lines. GAL1 protein expression was also uniformly high in HL cell lines and low or undectable in DLBCL lines by western blotting. Immunohistochemical staining of primary tumors revealed abundant GAL1 expression in HL RS cells whereas DLBCLs were uniformly negative. To elucidate the mechanisms responsible for GAL1 over expression in HL, we analyzed the GAL1 locus and identified a candidate AP1 enhancer element 1.5 kb downstream of the GAL1 transcription start site. The predicted enhancer element increased the expression of a reporter gene (luciferase) 5-fold in a HL cell line but had no effect in either a DLBCL or fibroblast cell line. In electrophoretic mobility studies, AP1 bound to the predicted GAL1 enhancer element in a cell-type specific manner. In addition, mutation of the AP1 binding site in the GAL1 enhancer abolished its activity. Since the AP1 components, cJUN and JUN-B, are known to be overexpressed in HL, we analyzed cJUN and JUN-B copy numbers and transcript abundance in the HL cell lines using 100K high-density SNP arrays and oligonucleotide RNA microarrays. We found increased copy numbers of cJUN and/or JUN-B in all HL cell lines tested and confirmed these findings using fluorescence in situ hybridization (FISH). Increased cJUN and JUN-B copy numbers were associated with significantly higher transcript abundance in the HL cell lines. In additional functional assays, we confirmed previous reports that recombinant GAL1 induced the apoptosis of CD3+ PHA-stimulated lymphoblasts in a dose- and time-dependent manner. Taken together, these studies indicate that HL RS cells selectively over express GAL1, at least in part, via AP1-mediated induction of the GAL1 enhancer. Given the profound effects of GAL1 on cytotoxic T cells, RS-specific expression of GAL1 likely inhibits the host anti-tumor response in HL.

Differential Expression of Activator Protein-1 Proteins in the Pineal Gland of Syrian Hamster and Rat May Explain Species Diversity in Arylalkylamine N-Acetyltransferase Gene Expression

Species differences have been reported for the nighttime regulation of arylalkylamine N-acetyltransferase (AA-NAT), the melatonin rhythm-generating enzyme. In particular, de novo synthesis of stimulatory transcription factors is required for Aa-nat transcription in the Syrian hamster but not in the rat pineal gland. The present work investigated the contribution of phosphorylated cAMP-responsive element-binding protein, c-FOS, c-JUN, and JUN-B in the regulation of Aa-nat transcription in Syrian hamsters compared with rats. The nighttime pattern of cAMP-responsive element-binding protein phosphorylation and regulation by norepinephrine observed in the Syrian hamster was similar to those reported in the rat. On the contrary, strong divergences in c-FOS, c-JUN, and JUN-B expression were observed between both species. In Syrian hamster, predominant expression of c-FOS and c-JUN was observed at the beginning of night, whereas a predominant expression of c-JUN and JUN-B was observed in the late night in rat. The early peak of c-FOS and c-JUN, known to form a stimulatory transcription dimer, suggests that they are involved in the nighttime stimulation of Aa-nat transcription. Indeed, early-night administration of a protein synthesis inhibitor (cycloheximide) markedly decreased AA-NAT mRNA levels in Syrian hamster. In the rat, high levels of JUN-B and c-JUN, constituting an inhibitory transcription dimer, are probably involved in the late-night inhibition of Aa-nat transcription. Early-night administration of cycloheximide actually increased AA-NAT mRNA levels toward the late night. Therefore, composition and timing of the pineal activator protein-1 complexes differ between rat and Syrian hamster and may be an activator (Syrian hamster) or an inhibitor (rat) of Aa-nat transcription.