TGF-ß-induced epigenetic deregulation of SOCS3 facilitates STAT3 signaling to promote fibrosis.

Fibroblasts are key effector cells in tissue remodeling. They remain persistently activated in fibrotic diseases, resulting in progressive deposition of extracellular matrix. Although fibroblast activation may be initiated by external factors, prolonged activation can induce an "autonomous," self-maintaining profibrotic phenotype in fibroblasts. Accumulating evidence suggests that epigenetic alterations play a central role in establishing this persistently activated pathologic phenotype of fibroblasts. We demonstrated that in fibrotic skin of patients with systemic sclerosis (SSc), a prototypical idiopathic fibrotic disease, TGF-ß induced the expression of DNA methyltransferase 3A (DNMT3A) and DNMT1 in fibroblasts in a SMAD-dependent manner to silence the expression of suppressor of cytokine signaling 3 (SOCS3) by promoter hypermethylation. Downregulation of SOCS3 facilitated activation of STAT3 to promote fibroblast-to-myofibroblast transition, collagen release, and fibrosis in vitro and in vivo. Reestablishment of the epigenetic control of STAT3 signaling by genetic or pharmacological inactivation of DNMT3A reversed the activated phenotype of SSc fibroblasts in tissue culture, inhibited TGF-ß-dependent fibroblast activation, and ameliorated experimental fibrosis in murine models. These findings identify a pathway of epigenetic imprinting of fibroblasts in fibrotic disease with translational implications for the development of targeted therapies in fibrotic diseases.

Poly(ADP-ribose) polymerase-1 regulates fibroblast activation in systemic sclerosis.

OBJECTIVES: The enzyme poly(ADP-ribose) polymerase-1 (PARP-1) transfers negatively charged ADP-ribose units to target proteins. This modification can have pronounced regulatory effects on target proteins. Recent studies showed that PARP-1 can poly(ADP-ribosyl)ate (PARylate) Smad proteins. However, the role of PARP-1 in the pathogenesis of systemic sclerosis (SSc) has not been investigated. METHODS: The expression of PARP-1 was determined by quantitative PCR and immunohistochemistry. DNA methylation was analysed by methylated DNA immunoprecipitation assays. Transforming growth factor-ß (TGFß) signalling was assessed using reporter assays, chromatin immunoprecipitation assays and target gene analysis. The effect of PARP-1 inactivation was investigated in bleomycin-induced and topoisomerase-induced fibrosis as well as in tight-skin-1 (Tsk-1) mice. RESULTS: The expression of PARP-1 was decreased in patients with SSc, particularly in fibroblasts. The promoter of PARP-1 was hypermethylated in SSc fibroblasts and in TGFß-stimulated normal fibroblasts. Inhibition of DNA methyltransferases (DNMTs) reduced the promoter methylation and reactivated the expression of PARP-1. Inactivation of PARP-1 promoted accumulation of phosphorylated Smad3, enhanced Smad-dependent transcription and upregulated the expression of TGFß/Smad target genes. Inhibition of PARP-1 enhanced the effect of TGFß on collagen release and myofibroblast differentiation in vitro and exacerbated experimental fibrosis in vivo. PARP-1 deficiency induced a more severe fibrotic response to bleomycin with increased dermal thickening, hydroxyproline content and myofibroblast counts. Inhibition of PARylation also exacerbated fibrosis in Tsk-1 mice and in mice with topoisomerase-induced fibrosis. CONCLUSION: PARP-1 negatively regulates canonical TGFß signalling in experimental skin fibrosis. The downregulation of PARP-1 in SSc fibroblasts may thus directly contribute to hyperactive TGFß signalling and to persistent fibroblast activation in SSc.

The histone demethylase Jumonji domain-containing protein 3 (JMJD3) regulates fibroblast activation in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis. METHODS: JMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP. RESULTS: The expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFß)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFß. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2. CONCLUSION: We present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models.

Diagnosis Threat and Injury Beliefs After Mild Traumatic Brain Injury.

BACKGROUND: Diagnosis threat is a psychosocial factor proposed to contribute to poor cognitive outcomes following mild traumatic brain injury (mTBI). The current research explored diagnosis threat impact on objective and subjective cognitive performance in a "high risk" population of athletes. Two possible moderators of diagnosis threat - injury beliefs and suggestibility - were also investigated. METHOD: Seventy-six participants with a history of mTBI were recruited through sports clubs and randomized to a months threat group (instructions drew attention to mTBI history) or a control group (no mention of mTBI). They completed a battery of neuropsychological tests and questionnaires regarding day-to-day cognitive abilities. Measures of depression, anxiety, illness beliefs and suggestibility were also collected. RESULTS: No significant group differences were found on any neuropsychological tasks, nor on self-report of cognitive difficulties. Illness beliefs were not found to play a moderating role in general, although the majority of the study sample did not report negative mTBI beliefs and expectations: concern about the consequences of injury was associated with weaker performance on one test, WAIS-III Digit Span performance. Suggestibility was also found to have a significant affect on this test. CONCLUSIONS: Diagnosis threat did not appear to have a marked affect on objective or subjective cognitive performance after mTBI in athletes. Differing injury beliefs between the study's athlete population and the general population is a possible explanation for different findings in the area. This and other sources of potential variation in the affect of diagnosis threat are discussed.

Randomised, waiting list controlled trial of cognitive-behavioural therapy for persistent postconcussional symptoms after predominantly mild-moderate traumatic brain injury.

BACKGROUND: Persistent postconcussional symptoms (PCS) can be a source of distress and disability following traumatic brain injury (TBI). Such symptoms have been viewed as difficult to treat but may be amenable to psychological approaches such as cognitive-behavioural therapy (CBT). OBJECTIVES: To evaluate the effectiveness of a 12-session individualised, formulation-based CBT programme. METHOD: Two-centre randomised waiting list controlled trial with 46 adults with persistent PCS after predominantly mild-to-moderate TBI (52% with post-traumatic amnesia (PTA)≤24 hours), but including some with severe TBIs (20% with PTA>7 days). RESULTS: Improvements associated with CBT were found on the primary outcome measures relating to quality of life (using the Quality of Life Assessment Schedule and the Brain Injury Community Rehabilitation Outcome Scale). Treatment effects after covarying for treatment duration were also found for PCS and several secondary outcomes, including measures of anxiety and fatigue (but not depression or post-traumatic stress disorder (PTSD)). Improvements were more apparent for those completing CBT sessions over a shorter period of time, but were unrelated to medicolegal status, injury severity or length of time since injury. CONCLUSIONS: This study suggests that CBT can improve quality of life for adults with persistent PCS and potentially reduce symptoms for some, in the context of outpatient brain injury rehabilitation services. TRIAL REGISTRATION NUMBER: ISRCTN49540320.