TAK1 Mediates TGF beta-1 Responses in Gingival Fibroblasts

In fibroblasts, transforming growth factor β-1 (TGFβ1) signals via canonical and non-canonical pathways to promote wound healing or hyper-contractile responses resulting in scars. The oral cavity however does not scar and fibrotic responses such as gingival hyperplasia are characterized by a hyper-proliferative response. The involvement of non-canonical, TGFβ-activated kinase-1 (TAK1) - mediated TGFβ1 signaling in gingival fibroblasts has not been previously examined. Here I show that TAK1 selective inhibitor (5Z)-7-Oxozeaenol blocks TGFβ1-induced expression of wound healing and fibrotic marker CCN2 (connective tissue growth factor) in gingival fibroblasts. Genome-wide expression profiling revealed that essentially all TGFβ1 induced genes were in fact sensitive to TAK1 inhibition in gingival fibroblasts (139/147) including those involved in proliferative and wound healing responses. This data was confirmed using RT-qPCR to detect mRNA expression as well as functional cell proliferation assay. (5Z)-7-Oxozeaenol might be investigated in the treatment of gingival hyperplasia in the future

Inhibition of cyclooxygenase activity by diclofenac attenuates varicose remodeling of mouse veins

Aim: The development of varicose veins is driven by risk factors that support the progression of venous hypertension, specifically, by chronically augmenting the circumferential tension of the venous wall. We have previously verified the relevance of this biomechanical stimulus for the activation of venous cells and the structural remodeling of the vein wall. Recent transcriptome analyses revealed an increase in the expression of the gene encoding prostaglandin-endoperoxide synthase 2 [cyclooxygenase 2 (COX-2)] in biomechanically stressed human vein endothelial cells. This observation prompted us to investigate the functional relevance of COX activity for the onset of pressure-induced venous remodeling.Methods: For the in vitro experiments, isolated mouse veins were exposed to elevated intraluminal pressure levels to study the markers of cellular activation. For the in vivo experiments, pressure-dependent varicose remodeling of veins was induced by ligation of an efferent vein in the mouse auricle. Diclofenac was applied to inhibit the activity of COX.Results: Short-term exposure to elevated pressure levels stimulated the abundance of activated matrix-metalloproteinase-2 (MMP-2) and mitogen activated protein kinase, ERK1/2, in isolated mouse veins, which was inhibited upon treatment with diclofenac. Transdermal application of diclofenac-containing phospholipid-micelles attenuated the corkscrew-like enlargement of veins and decreased the abundance of COX-2 and MMP-2 as well as cell proliferation in the venous wall.Conclusion: The cyclooxygenase inhibitor, diclofenac, interferes with stress-induced activation of venous cells and attenuates venous remodeling in vivo. Additional research is warranted to investigate whether nonsteroidal anti-inflammatory drugs interfere with the processes promoting the onset of varicose vein development and biomechanical activation of venous cells

Glycyrrhetinic Acid Antagonizes Pressure-Induced Venous Remodeling in Mice

Development of spider veins is caused by the remodeling of veins located in the upper dermis and promoted by risk factors such as obesity or pregnancy that chronically increase venous pressure. We have repeatedly shown that the pressure-induced increase in biomechanical wall stress is sufficient to evoke the formation of enlarged corkscrew-like superficial veins in mice. Subsequent experimental approaches revealed that interference with endothelial- and/or smooth muscle cell (SMC) activation counteracts this remodeling process. Here, we investigate whether the herbal agent glycyrrhetinic acid (GA) is a suitable candidate for that purpose given its anti-proliferative as well as anti-oxidative properties. While basic abilities of cultured venous SMCs such as migration and proliferation were not influenced by GA, it inhibited proliferation but not angiogenic sprouting of human venous endothelial cells (ECs). Further analyses of biomechanically stimulated ECs revealed that GA inhibits the DNA binding capacity of the mechanosensitive transcription factor activator protein-1 (AP-1) which, however, had only a minor impact on the endothelial transcriptome. Nevertheless, by decreasing gelatinase activity in ECs or mouse veins exposed to biomechanical stress, GA diminished a crucial cellular response in the context of venous remodeling. In line with the observed inhibitory effects, local transdermal application of GA attenuated pressure-mediated enlargement of veins in the mouse auricle. In summary, our data identifies GA as an inhibitor of EC proliferation, gelatinase activity and venous remodeling. It may thus have the capacity to attenuate spider vein formation and remodeling in humans

Cluster analysis of TAK1 depended mRNAs with over 1.7 fold induction (average of two arrays) in response to TGFβ-1 treatment.

<p>Genes involved with a hyperproliferative response are shown.</p><p>Cluster analysis of TAK1 depended mRNAs with over 1.7 fold induction (average of two arrays) in response to TGFβ-1 treatment.</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced CCN2 mRNA expression in human gingival fibroblasts.

<p>Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with or without TGFβ1 (4ngml^-1). Total RNA was harvested six hours later and subjected to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. Values are expressed relative to untreated control. (N = 3; averages+/-SEM are shown; **** = p<0.0001, * = p<0.05 One-Way ANOVA).</p

(5Z)-7-oxozeaenol inhibits TGFβ1-induced mRNA expression in human gingival fibroblasts.

<p>(A) Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with TGFβ1 (4ngml^-1 (90 pM)) ligand or left untreated. Total RNA was harvested six hours later and subjected to gene expression profiling using GeneChip Human Gene 1.0 ST arrays (N = 2) as described in Methods. 147 genes were up-regulated in response to TGFβ1 (1.7 fold induction compared to DMSO control group) and 139 genes of the latter group were found to be (5<i>Z</i>)-7-Oxozeaenol sensitive. (B) Human gingival fibroblasts were treated as in (A) and subject to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. (N = 3; averages+/-SEM are shown. * = p<0.05; ** = p<0.01; *** = p<0.001, One-Way ANOVA).</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced CCN2 protein expression in human gingival fibroblasts.

<p>(A) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-CCN2 and anti-β-actin antibodies, as indicated. A representative blot is shown. Experiments were performed on 4 separate occasions and relative CCN2 expression in response to TGFβ1 was calculated using densitometry (N = 4, averages+/-SEM are shown; * = p<0.05, Student’s t-test. CCN2 expression in response to TGFβ was taken to represent 1). (B) Indirect immunofluorescence analysis. Human gingival fibroblasts cultured on glass coverslips as treated as in (A). Cells were fixed and stained with an anti-CCN2 antibody and DyLight 594 conjugated secondary antibody. Cells were counterstained with DAPI to detect nuclei. Representative photographs are shown. Experiments were conducted four times, and relative fluoresce intensity ratio was calculated as described in methods (N = 4, averages+/-SEM are shown. * = p<0.05, One-Way ANOVA). (C) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-phospho-TAK1 and anti-beta actin antibodies, as indicated.</p

(5Z)-7-oxozeaenol reduces TGFβ1 induced gingival fibroblast proliferation.

<p>Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol ((5Z)-7-oxo; 400 nM) or DMSO for 45 min followed by treatment with TGFβ1 (4ngml-1 (90 pM)) ligand or left untreated. Cultures were grown in the presence of BrdU for up to 72 hours as described in methods. One of three representative experiments is shown; (N = 4; averages+/-SEM are shown * p<0.05 for: DMSO vs TGFβ1, (5Z)-7-oxo vs TGFβ1, TGFβ1 vs (5Z)-7-oxo+TGFβ1; ** p<0.05 for: DMSO vs TGFβ1; (5Z)-7-oxo vs TGFβ1, TGFβ1 vs (5Z)-7-oxo+TGFβ1. Two-Way ANOVA followed by Tukey's Post Hoc analysis).</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced collagen expression.

<p>A) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-collagen type I and anti-β-actin antibodies, as indicated. A representative blot is shown. Experiments were performed on 3 separate occasions. (N = 4, averages+/-SEM are shown; * = p<0.05, Student’s t-test. CCN2 expression in response to TGFβ was taken to represent 1). (B) mRNA analysis Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with or without TGFβ1 (4ngml^-1 (90 pM)). Total RNA was harvested six hours later and subjected to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. (N = 3; averages+/-SEM are shown; * = p<0.05, One-Way ANOVA).</p