15 research outputs found
MotomiRs: miRNAs in Motor Neuron Function and Disease
MiRNAs are key regulators of the mammalian transcriptome that have been increasingly linked to degenerative diseases of the motor neurons. Although many of the miRNAs currently incriminated as participants in the pathogenesis of these diseases are also important to the normal development and function of motor neurons, at present there is no knowledge of the complete miRNA profile of motor neurons. In this review, we examine the current understanding with respect to miRNAs that are specifically required for motor neuron development, function and viability, and provide evidence that these should be considered as a functional network of miRNAs which we have collectively termed MotomiRs. We will also summarize those MotomiRs currently known to be associated with both amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), and discuss their potential use as biomarkers
RECK-Mediated β1-Integrin Regulation by TGF-β1 Is Critical for Wound Contraction in Mice
<div><p>Fibroblasts are critical for wound contraction; a pivotal step in wound healing. They produce and modify the extracellular matrix (ECM) required for the proper tissue remodeling. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a key regulator of ECM homeostasis and turnover. However, its role in wound contraction is presently unknown. Here we describe that Transforming growth factor type β1 (TGF-β1), one of the main pro-fibrotic wound-healing promoting factors, decreases RECK expression in fibroblasts through the Smad and JNK dependent pathways. This TGF-β1 dependent downregulation of RECK occurs with the concomitant increase of β1-integrin, which is required for fibroblasts adhesion and wound contraction through the activation of focal adhesion kinase (FAK). Loss and gain RECK expression experiments performed in different types of fibroblasts indicate that RECK downregulation mediates TGF-β1 dependent β1-integrin expression. Also, reduced levels of RECK potentiate TGF-β1 effects over fibroblasts FAK-dependent contraction, without affecting its cognate signaling. The above results were confirmed on fibroblasts derived from the <i>Reck</i><sup>+/-</sup> mice compared to wild type-derived fibroblasts. We observed that <i>Reck</i><sup>+/-</sup> mice heal dermal wounds more efficiently than wild type mice. Our results reveal a critical role for RECK in skin wound contraction as a key mediator in the axis: TGF-β1—RECK- β1-integrin.</p></div
Downregulation of RECK in fibroblasts is required for TGF-β1 induced matrix contraction.
<p><b>(A)</b> Schematic of the matrix contraction assay. From top to bottom, fibroblasts were mixed with a collagen solution that polymerizes into a 3D collagen matrix for 2 h. This mixture was then released from the culture dish and allowed to float in culture medium. Floating collagen lattices were incubated with TGF-β1 for 24 hours to induce fibroblasts contraction, leading to a volume reduction of the collagen lattice. <b>(B)</b> NIH3T3 fibroblasts were transiently transfected with a pool of shRNAs against mouse RECK (shReck) or with a RECK overexpression vector or with an empty vector as control. 24 h post-transfection, fibroblasts were trypsinized and a collagen contraction assay was performed as described in (A). After 24 h, the now trypsinized collagen lattices were treated with TGF-β1 and/or the focal adhesion kinase inhibitor 14 (FAK-Inh) for 24 h. Afterwards, 3D floating matrices were photographed. Representative images are shown. <b>(C)</b> The volume of the contracted matrices obtained in (B) was measured immediately after being released at the end of the assay. These values, obtained from two independent experiments performed in triplicate, were graphed as a percentage of the initial volume. Statistical significance was assessed using two-way ANOVA and a Bonferroni multiple-comparison post hoc test. &, P<0.05 relative to TGF-β1 untreated Empty vector; #, P<0.05 relative to TGF-β1 untreated RECK; ¢, P<0.05 relative to TGF-β1 treated Empty vector; ∞, P<0.05 relative to TGF-β1 treated RECK. <b>(D)</b> Aliquots of the trypsinized cells in (B) were directly seeded in culture dishes before mixing with the collagen solution and then treated with TGF-β1 and/or the FAK-Inh for 24 has described in (B). Cell extracts were prepared and analyzed by Western blot to determine the levels of RECK and β1-integrin. Tubulin levels were used as a loading control.</p
TGF-β1 decreases RECK expression in fibroblasts.
<p><b>(A)</b> NIH3T3 fibroblasts and were incubated with TGF-β1 for 24 and 48 h at the indicated concentrations. Western blots analysis of cell extracts were performed to determine the expression of RECK, β1-integrin, FN and CTGF. β-tubulin (Tubulin) levels were used as a loading control. <b>(B)</b> Primary fibroblasts cultures derived from tibialis anterior skeletal muscles (SM) and skin biopsies of 3-month-old WT mice were incubated with TGF-β1 for 24 as indicated. Western blot analysis of cell extracts were performed to determine the levels of RECK, FN, and CTGF. tubulin levels were used as a loading control. <b>(C)</b> NIH3T3 fibroblasts were incubated with 5 ng/ml of TGF-β1 for the indicated times. At the end of the assay, total RNA was extracted and was reverse transcribed into complementary DNA. Taqman quantitative real-time PCR was performed to determine <i>Reck</i> expression. mRNA expression was quantified using the comparative ΔC<sub>T</sub> method (2<sup>-ΔΔCT</sup>) using <i>Gadph</i> as a reference gene. mRNA levels are presented relative to the mean expression of the control (untreated cells). <b>(D)</b><i>Reck</i> mRNA expression in NIH3T3 fibroblasts incubated with different concentrations of TGF-β1 for 24 hours was determined as in (C). In C and D, values are expressed as mean +/- standard deviation (SD) of two independent experiments. In C, &, P<0.05 relative to 0 hour; #, P<0.05 relative to 6 hours. In D, &, P<0.05 relative to 0 ng/ml; #, P<0.05 relative to 0,5 ng/ml.</p
<i>Reck</i> deficient mice have accelerated skin wound contraction.
<p>A dermal punch wound contraction assay on the back of 3- month-old WT and <i>Reck</i><sup><i>+/-</i></sup> mice was performed. The wounds were photographed at the moment of the surgery (D0) and after 3 (D3) and 7 (D7) days. Representative photographs are shown in <b>(A)</b>. The wounded area was determined at D0, D3 and D7 and expressed as percentage of the initial wound area, as shown in <b>(B)</b>. Values correspond to mean ± S.D. of two independent experiments, using four mice for each experimental condition. Statistical significance was assessed using two-way ANOVA and a Bonferroni multiple-comparison post hoc test. &, P<0.05 relative to WT D3; #, P<0.05 relative to WT D7; ¢, P<0.05 relative <i>RECK+/-</i> D3.</p
TGF-β1 decreases RECK levels through a Smad and JNK dependent <i>pathway</i>.
<p><b>(A)</b> NIH3T3 fibroblasts were pre-treated for 30 minutes with different inhibitors: TGF-β-RI kinase inhibitor SB525334, PI3K inhibitor LY294002, MEK1 inhibitor PD98059, JNK inhibitor SB600125, and p38 inhibitor SB203580. After the pre-treatment, fibroblasts were treated with 5ng/ml of TGF-β1 for 24 hours or untreated to serve as controls. Western blot analysis of cell extracts were performed to determine the levels of RECK and FN. tubulin levels were used as a loading control. <b>(B)</b> NIH3T3 fibroblasts were transiently transfected with a pool of siRNA against Smad-2 and Smad-3 or with a scrambled siRNA as a control. 24 h post-transfection, cells were treated with 5 ng/ml of TGF-β1 for 24 h, or left untreated to serve as a control. Western blot analysis of cell extracts were performed to determine the levels of RECK, Smad-3 and β1-Integrin. β-tubulin levels were used as a loading control. <b>(C)</b> NIH3T3 fibroblasts were pre-treated for 30 minutes with SIS3, a specific inhibitor of Smad-3 activation, and the JNK inhibitor SB600125; cells were treated either alone or in combination. Afterwards, fibroblasts were treated with 5 ng/ml of TGF-β1, or left untreated as a control, for 24h. Western blot analysis of cell extracts were performed to determine the levels of RECK and β1-Integrin. β-tubulin levels were used as a loading control. The quantifications shown in A, B and C are from two independent. Statistical significance was assessed using two-way ANOVA and a Bonferroni multiple-comparison post hoc test. &, P<0.05 relative to TGF-β1 untreated fibroblasts.</p
<i>Reck</i><sup>+/-</sup> fibroblasts show increased level of β1-integrin and enhanced contractile properties.
<p><b>(A)</b> Primary fibroblasts cultures derived from skin biopsies of 3 month old WT (skin-Fbs<sup>WT</sup>) and <i>Reck</i><sup><i>+/-</i></sup> (skin-Fbs<sup>Reck+/-</sup>) mice were incubated with TGF-β1 for 24 hours as indicated. Western blot analysis of cell extracts were performed to determine the levels of RECK, β1-integrin and CTGF. Tubulin levels were used as a loading control. <b>(B)</b> Quantification of the expression of RECK and β1-integrin relative to tubulin expression. values are expressed as mean +/- SD of two independent experiments. &, P<0.05 relative to TGF-β1 untreated skin-Fbs<sup>WT</sup>; #, P<0.05 relative to TGF-β1 untreated skin-Fbs<sup>Reck+/-</sup>. <b>(C)</b> A collagen contraction assay using primary culture fibroblasts derived from skin biopsies of 3 month old WT (skin-Fbs<sup>WT</sup>) and <i>Reck</i><sup><i>+/-</i></sup> (skin-Fbs<sup>Reck+/-</sup>) mice was performed. The floating collagen lattices were treated with TGF-β1 (3 ng/ml). After 24 hours of treatment, the 3D floating matrices were photographed. Representative images are shown. <b>(D)</b> The volumes of the contracted matrices obtained in (A) were measured immediately after being released at the end of the assay. The values, obtained from two independent experiments performed in triplicate, were graphed as a percentage of the initial volume. Statistical significance was assessed using two-way ANOVA and a Bonferroni multiple-comparison post hoc test. &, P<0.05 relative to TGF-β1 untreated skin-Fbs<sup>WT</sup>; #, P<0.05 relative to TGF-β1 untreated skin-Fbs<sup>Reck+/</sup>; ¢, P<0.05 relative to TGF-β1 treated skin-Fbs<sup>WT</sup>.</p
TGF-β1 must reduce RECK levels to increase β1-integrin levels in fibroblasts.
<p><b>(A)</b> NIH3T3 fibroblasts were transiently transfected with a pool of shRNAs against mouse RECK (shRECK) or with a scrambled sequence as control (shCtrl). 24 h post-transfection, cells were treated or not with 5 ng/ml of TGF-β1 for 24 h (<b>B</b>) NIH3T3 fibroblasts were transiently transfected with a vector that overexpresses RECK or with an empty vector as control. 24 h post-transfection, cells were treated with 5 ng/ml of TGF-β1 for 24 h, or left untreated as a control. In A and B, Western blot analysis of cell extracts were performed to determine the levels of RECK, β1-integrin and CTGF. Tubulin levels were used as a loading control. In A, &, P<0.05 relative to untreated shCtrl; #, P<0.05 relative to untreated shRECK. In B, &, P<0.05 relative to TGF-β1 untreated Empty vector; #, P<0.05 relative to TGF-β1 untreated RECK; ¢, P<0.05 relative to TGF-β1 treated Empty vector.</p