Catechol-O-methyltransferase (COMT) is a key regulator of pain perception, cognitive function, and affective mood. Three common haplotypes of the humanCOMTgene, divergent in two synonymous and one nonsynonymous position, code for differences in COMT enzymatic activity and are associated with pain sensitivity. Haplotypes divergent in synonymous changes exhibited the largest difference in COMT enzymatic activity, due to a reduced amount of translated protein. The majorCOMThaplotypes varied with respect to messenger RNA local stem-loop structures, such that the most stable structure was associated with the lowest protein levels and enzymatic activity. Site-directed mutagenesis that eliminated the stable structure restored the amount of translated protein. These data highlight the functional significance of synonymous variations and suggest the importance of haplotypes over single-nucleotide polymorphisms for analysis of genetic variations.

Growth differentiation factor-9 (GDF-9) is an oocyte-derived growth factor and a member of the TGF-beta superfamily that includes TGF-beta, activin, and bone morphogenetic proteins (BMPs). GDF-9 is indispensable for the development of ovarian follicles from the primary stage, and treatment with GDF-9 enhances the progression of early follicles into small preantral follicles. Similar to other TGF-beta family ligands, GDF-9 likely initiates signaling mediated by type I and type II receptors with serine/threonine kinase activity, followed by the phosphorylation of intracellular transcription factors named Smads. We have shown previously that GDF-9 interacts with the BMP type II receptor (BMPRII) in granulosa cells, but the type I receptor involved is unknown. Using P19 cells, we now report that GDF-9 treatment stimulated the CAGA-luciferase reporter known to be responsive to TGF-beta mediated by the type I receptor, activin receptor-like kinase (ALK)5. In contrast, GDF-9 did not stimulate BMP-responsive reporters. In addition, treatment with GDF-9 induced the phosphorylation of Smad2 and Smad3 in P19 cells, and the stimulatory effect of GDF-9 on the CAGA-luciferase reporter was blocked by the inhibitory Smad7, but not Smad6. We further reconstructed the GDF-9 signaling pathway using Cos7 cells that are not responsive to GDF-9. After overexpression of ALK5, with or without exogenous Smad3, the Cos7 cells gained GDF-9 responsiveness based on the CAGA-luciferase reporter assay. The roles of ALK5 and downstream pathway genes in mediating GDF-9 actions were further tested in ovarian cells. In cultured rat granulosa cells from early antral follicles, treatment with GDF-9 stimulated the CAGA-luciferase reporter activity and induced the phosphorylation of Smad3. Furthermore, transfection with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 actions. In conclusion, although GDF-9 binds to the BMP-activated type II receptor, its downstream actions are mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins. Because ALK5 is a known receptor for TGF-beta, diverse members of the TGF-beta family of ligands appear to interact with a limited number of receptors in a combinatorial manner to activate two downstream Smad pathways.

Background Histone deacetylase inhibitors (HDACi) display potent therapeutic efficacy in animal models of arthritis and suppress inflammatory cytokine production in rheumatoid arthritis (RA) synovial macrophages and tissue. Objectives To determine the molecular mechanisms contributing to the suppressive effects of HDACi on RA synovial cell activation, using interleukin 6 (IL-6) regulation as a model. Methods RA fibroblast-like synoviocytes (FLS) and healthy donor macrophages were treated with IL-1β, tumour necrosis factor (TNF)α, lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C)) in the absence or presence of the HDACi trichostatin A (TSA) or ITF2357 (givinostat). IL-6 production and mRNA expression was measured by ELISA and quantitative PCR (qPCR), respectively. Protein acetylation and the activation of intracellular signalling pathways were assessed by immunoblotting. The DNA-binding activity of nuclear factor κB (NFκB) and activator protein 1 (AP-1) components was measured by ELISA-based assays. Results HDACi (0.25–1.0 μM) suppressed RA FLS IL-6 production induced by IL-1β, TNFα and Toll-like receptor ligands. Phosphorylation of mitogen-activated protein kinases and inhibitor of κBα (IκBα) following IL-1β stimulation were unaffected by HDACi, as were AP-1 composition and binding activity, and c-Jun induction. TSA induced a significant reduction in nuclear retention of NFκB in FLS 24 h after IL-1β stimulation, but this did not reduce NFκB transcriptional activity or correlate temporally with reductions in IL-6 mRNA accumulation. HDACi significantly reduced the stability of IL-6 mRNA in FLS and macrophages. Conclusions Our study identifies a novel, shared molecular mechanism by which HDACi can disrupt inflammatory cytokine production in RA synovial cells, namely the promotion of mRNA decay, and suggests that targeting HDAC activity may be clinically useful in suppressing inflammation in RA.

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.

Cell-cycle exit and differentiation of suprabasal epidermal keratinocytes require nuclear IκB kinase α (IKKα), but not its protein kinase activity. IKKα also is a suppressor of squamous cell carcinoma (SCC), but its mode of action remains elusive. Postulating that IKKα may serve as a transcriptional regulator in keratinocytes, we searched for cell-cycle-related genes that could illuminate this function. IKKα was found to control several Myc antagonists, including Mad1 , Mad2 , and Ovol1 , through the association with TGFβ-regulated Smad2/3 transcription factors and is required for Smad3 recruitment to at least one of these targets. Surprisingly, Smad2/3-dependent Mad1 induction and keratinocyte differentiation are independent of Smad4, the almost universal coregulator of canonical TGFβ signaling. IKKα also is needed for nuclear accumulation of activated Smad2/3 in the epidermis, and Smad2/3 are required for epidermal differentiation. We suggest that a TGFβ–Smad2/3–IKKα axis is a critical Smad4-independent regulator of keratinocyte proliferation and differentiation.