Ets-1 Is Essential for Connective Tissue Growth Factor (CTGF/CCN2) Induction by TGF-β1 in Osteoblasts

Ets-1 controls osteoblast differentiation and bone development; however, its downstream mechanism of action in osteoblasts remains largely undetermined. CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts; however, the molecular mechanisms that control CCN2 induction are poorly understood. In this study, we investigated the role of Ets-1 for CCN2 induction by TGF-β1 in primary osteoblasts.We demonstrated that Ets-1 is expressed and induced by TGF-β1 treatment in osteoblasts, and that Ets-1 over-expression induces CCN2 protein expression and promoter activity at a level similar to TGF-β1 treatment alone. Additionally, we found that simultaneous Ets-1 over-expression and TGF-β1 treatment synergize to enhance CCN2 induction, and that CCN2 induction by TGF-β1 treatment was impaired using Ets-1 siRNA, demonstrating the requirement of Ets-1 for CCN2 induction by TGF-β1. Site-directed mutagenesis of eight putative Ets-1 motifs (EBE) in the CCN2 promoter demonstrated that specific EBE sites are required for CCN2 induction, and that mutation of EBE sites in closer proximity to TRE or SBE (two sites previously shown to regulate CCN2 induction by TGF-β1) had a greater effect on CCN2 induction, suggesting potential synergetic interaction among these sites for CCN2 induction. In addition, mutation of EBE sites prevented protein complex binding, and this protein complex formation was also inhibited by addition of Ets-1 antibody or Smad 3 antibody, demonstrating that protein binding to EBE motifs as a result of TGF-β1 treatment require synergy between Ets-1 and Smad 3.This study demonstrates that Ets-1 is an essential downstream signaling component for CCN2 induction by TGF-β1 in osteoblasts, and that specific EBE sites in the CCN2 promoter are required for CCN2 promoter transactivation in osteoblasts

Increased Serum and Musculotendinous Fibrogenic Proteins following Persistent Low-Grade Inflammation in a Rat Model of Long-Term Upper Extremity Overuse.

We examined the relationship between grip strength declines and muscle-tendon responses induced by long-term performance of a high-repetition, low-force (HRLF) reaching task in rats. We hypothesized that grip strength declines would correlate with inflammation, fibrosis and degradation in flexor digitorum muscles and tendons. Grip strength declined after training, and further in weeks 18 and 24, in reach limbs of HRLF rats. Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1β after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles. Similar cytokine increases were detected in serum with HRLF: IL-1α and IL-10 in week 18, and TNF-α and IL-6 in week 24. Grip strength correlated inversely with IL-6 in muscles, tendons and serum, and TNF-α in muscles and serum. Four fibrogenic proteins, TGFB1, CTGF, PDGFab and PDGFbb, and hydroxyproline, a marker of collagen synthesis, increased in serum in HRLF weeks 18 or 24, concomitant with epitendon thickening, increased muscle and tendon TGFB1 and CTGF. A collagenolytic gelatinase, MMP2, increased by week 18 in serum, tendons and muscles of HRLF rats. Grip strength correlated inversely with TGFB1 in muscles, tendons and serum; with CTGF-immunoreactive fibroblasts in tendons; and with MMP2 in tendons and serum. Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance. Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed

Weight and grip strength changes across weeks of task performance.

<p>(A) All rats gained weight across the 29 weeks. There were no significant differences in body weights between trained only (TR0 and TR24) or high repetition low force (HRLF) rats, compared to normal controls (NC) rats. (B) Maximum reflexive grip strength is shown for control rats (normal and food-restricted controls were combined as there were no significant differences between these groups), TR24 (TR+Rest; trained rats that rested after training for 24 weeks), and HRLF rats. The preferred reach limbs and the contralateral support limbs were analyzed separately in HRLF rats. The week 0 time point of each group is after the training period and before the HRLF task performance or the rest period. Symbols: *:p<0.05 and ***:p<0.001, compared to age-matched control rats; ^aa: p<0.01 and ^aaa:p<0.001, compared to age-matched TR24 rats; ^b: p<0.05, compared to the support limb of HRLF rats.</p

Design of experiment.

<p>All rats were handled daily for week 1. Then, all but normal control rats (NC) were food restricted to within 5% of the weights of the NC rats for the remainder of the experiments. Food restricted control rats (FRC) did not undergo training and did not perform the high repetition low force (HRLF) task. A cohort of rats were trained only for 4 weeks, and then euthanized (TR0). Another cohort was trained only for 4 weeks, and then rested for 24 weeks (TR24). Two groups of rats performed the HRLF task for either 18 weeks or 24 weeks (18-week HRLF and 24-week HRLF rats). After euthanasia, serum (S) was collected from all rats and assayed in nearly all rats. Half of the tissues were analyzed using biochemical methods (B) or histological methods (H).</p

Pro- and anti-inflammatory cytokines in serum.

<p>Groups are as defined in figure legend 2. Data is shown for (A) TNF-alpha, (B) IL-6, (C) IL-10, (D), IL-1alpha, (E) IL-12 (only TR24 and 18 wk HRLF rat serum were tested for IL-12), and (F) MIP2. Symbols: *:p<0.05, **:p<0.01, compared to age-matched control rats. ^a: p<0.05, compared to age-matched trained rats that rested for 24 weeks after the initial training period.</p

Transforming growth factor beta 1 (TGFB1) in serum and flexor digitorum tissues of the reach limbs.

<p>Groups are as defined in figure legend 2. (A&B) Serum TGFB1 and tendon TGFB1, assayed using ELISA. (C) Immunohistochemical staining for TGFB1 in tendons from NC and 18-week HRLF reach limbs shows localization of TGFB1 in fibroblast-like cells in the peritendon region of both the NC and HRLF rats, and additional stained cells in the epitendon (Epi; thickened in HRLF rats) and endotendon (T) regions of the HRLF rat tendon. (D&E) The results of Western blot analysis of muscle TGFB1 in which two bands were detected, 50 kDa and 12.5 kDa. The ratio of each band of TGFB1 normalized to GAPDH levels is shown for three replicates of the western blot. (F) A representative Western blot of reach limb muscles from normal controls (NC, n = 4 shown), 24-week HRLF rats (n = 3 shown), and 18-week HRLF rats (n = 4 shown), probed with anti-TGFB1 and GAPDH. Green bands were detected with an anti-TGFB1 antibody and a secondary antibody tagged with IRDye800CW (Li-Cor, #.926-32211). Red bands were detected with an anti-GAPDH antibody and a secondary antibody tagged IRDye680LT (Li-Cor, #926-68020). Symbols: *:p<0.05, **:p<0.01, compared to age-matched control rats; ^a: p<0.05, compared to TR24 rats. Scale bar = 50 micrometers.</p

Pro- and anti-inflammatory cytokines in forelimb flexor digitorum tissues.

<p>Data shown for cytokines assayed in muscles and tendons after collection from control rats (indicated as C; normal and food restricted control data was combined as there was no significant difference), trained only rats euthanized immediately after training ( indicated as TR0 on the x-axes), TR+Rest (TR24; trained rats that rested for 24 weeks; blue dashed lines), and high repetition low force (HRLF) rats that worked for 18 or 24 weeks of task performance before tissue collection. The preferred reach limbs (red line) and the contralateral support limbs (green lines) were examined separately in HRLF rats. (A & B) Muscle and Tendon IL-1beta. (C & D) Muscle and Tendon TNF-alpha. (E & F) Muscle and Tendon IL-6. (G & H) Muscle and Tendon IL-10. Each analyte was assayed in duplicate using single-plex ELISA kits. Symbols: *:p<0.05, **:p<0.01 and ***:p<0.001, compared to age-matched control rats; ^a: p<0.05 and ^aa:p<0.01, compared to age-matched TR+Rest rats; ^b: p<0.05, compared to the support limb of HRLF rats.</p

Platelet derived growth factor ab (PDGFab) and PDGF bb in serum and flexor digitorum tendons of the reach limbs, and hydroxyproline in serum and flexor digitorum muscles of the reach limbs.

<p>ELISA data for normal controls have been combined with that of food restricted controls (C); TR0 (trained only rats), TR21 (trained rats that rested for 21 weeks), and high repetition low force (HRLF) rats that performed the task for 18 or 24 weeks. ** and *: p<0.05 and p<0.01, compared to C rats; ^a: p<0.05, compared to TR0 rats.</p

MMP2 levels in serum and flexor digitorum tendons and muscles of the reach limbs.

<p>Groups are as defined in figure legend 2. MMP2 levels in (A) serum and (B) flexor digitorum tendons tested using ELISA. *:p<0.05, compared to C; ^a: p<0.05, compared to TR0 rats. (C) Gelatin zymography showing that muscle homogenates of 24-week HRLF rats have increased pro-MMP2 (72 kDa) and active MMP2 (62 kDa) compared to NC rats. Neither group showed any MMP9 activity. The far left lane is the marker (M). The far right lane was loaded with homogenized mouse spleen, which contains MMP9 and low levels of active MMP2.</p