scholarly journals Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes

2009 ◽  
Vol 297 (4) ◽  
pp. C898-C906 ◽  
Author(s):  
Simon R. Tew ◽  
Mandy J. Peffers ◽  
Tristan R. McKay ◽  
Emma T. Lowe ◽  
Wasim S. Khan ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Half Life ◽  
Articular Chondrocytes ◽  
Kinase Inhibitor ◽  
Stress Fibers ◽  
Human Articular Chondrocytes ◽  
Luciferase Assay ◽  
Mrna Levels ◽  
Matrix Gene ◽  
Actin Stress Fibers

The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471–39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2- 4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24–48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.

scholarly journals Effects of Netarsudil on Actin-Driven Cellular Functions in Normal and Glaucomatous Trabecular Meshwork Cells: A Live Imaging Study

2020 ◽  
Vol 9 (11) ◽  
pp. 3524
Author(s):  
Kate E. Keller ◽  
Casey Kopczynski
Keyword(s):  
Trabecular Meshwork ◽  
Kinase Inhibitor ◽  
Imaging Study ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Cellular Functions ◽  
Tunneling Nanotubes ◽  
Trabecular Meshwork Cells ◽  
Lateral Fusion ◽  
Actin Stress Fibers

The actin cytoskeleton of trabecular meshwork (TM) cells is a therapeutic target for lowering intraocular pressure (IOP) in glaucoma patients. Netarsudil (the active ingredient in RhopressaTM) is a Rho-associated protein kinase inhibitor that induces disassembly of actin stress fibers. Here, we used live cell imaging of SiR-actin-labeled normal (NTM) and glaucomatous TM (GTM) cells to investigate actin dynamics during actin-driven biological processes with and without netarsudil treatment. Actin stress fibers were thicker in GTM than NTM cells and took longer (>120 min) to disassemble following addition of 1 µM netarsudil. Actin-rich extracellular vesicles (EVs) were derived by two mechanisms: exocytosis of intracellular-derived vesicles, and cleavage of filopodial tips, which detached the filopodia from the substratum, allowing them to retract to the cell body. While some phagocytosis was noted in untreated TM cells, netarsudil potently stimulated phagocytic uptake of EVs. Netarsudil treatment induced lateral fusion of tunneling nanotubes (TNTs) that connected adjacent TM cells; TNTs are important for TM cellular communication. Together, our results suggest that netarsudil may clear outflow channels in TM tissue by inducing phagocytosis and/or by modulating TM communication via EVs and TNTs. These cellular functions likely work together to regulate IOP in normal and glaucomatous TM.

scholarly journals WT1-interacting protein (Wtip) regulates podocyte phenotype by cell-cell and cell-matrix contact reorganization

2012 ◽  
Vol 302 (1) ◽  
pp. F103-F115 ◽  
Author(s):  
Jane H. Kim ◽  
Amitava Mukherjee ◽  
Sethu M. Madhavan ◽  
Martha Konieczkowski ◽  
John R. Sedor
Keyword(s):  
Kinase Inhibitor ◽  
Adherens Junctions ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Transcriptional Responses ◽  
Interacting Protein ◽  
Cell Matrix ◽  
Actin Stress Fibers ◽  
Cell Cell

Podocytes respond to environmental cues by remodeling their slit diaphragms and cell-matrix adhesive junctions. Wt1-interacting protein (Wtip), an Ajuba family LIM domain scaffold protein expressed in the podocyte, coordinates cell adhesion changes and transcriptional responses to regulate podocyte phenotypic plasticity. We evaluated effects of Wtip on podocyte cell-cell and cell-matrix contact organization using gain-of- and loss-of-function methods. Endogenous Wtip targeted to focal adhesions in adherent but isolated podocytes and then shifted to adherens junctions after cells made stable, homotypic contacts. Podocytes with Wtip knockdown (shWtip) adhered but failed to spread normally. Noncontacted shWtip podocytes did not assemble actin stress fibers, and their focal adhesions failed to mature. As shWtip podocytes established cell-cell contacts, stable adherens junctions failed to form and F-actin structures were disordered. In shWtip cells, cadherin and β-catenin clustered in irregularly distributed spots that failed to laterally expand. Cell surface biotinylation showed diminished plasma membrane cadherin, β-catenin, and α-catenin in shWtip podocytes, although protein expression was similar in shWtip and control cells. Since normal actin dynamics are required for organization of adherens junctions and focal adhesions, we determined whether Wtip regulates F-actin assembly. Undifferentiated podocytes did not elaborate F-actin stress fibers, but when induced to overexpress WTIP, formed abundant stress fibers, a process blocked by the RhoA inhibitor C3 toxin and a RhoA kinase inhibitor. WTIP directly interacted with Rho guanine nucleotide exchange factor (GEF) 12 (Arhgef12), a RhoA-specific GEF enriched in the glomerulus. In conclusion, stable assembly of podocyte adherens junctions and cell-matrix contacts requires Wtip, a process that may be mediated by spatiotemporal regulation of RhoA activity through appropriate targeting of Arhgef12.

scholarly journals PKR Promotes Oxidative Stress and Apoptosis of Human Articular Chondrocytes by Causing Mitochondrial Dysfunction through p38 MAPK Activation—PKR Activation Causes Apoptosis in Human Chondrocytes

Antioxidants ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 370 ◽  
Author(s):  
Ching-Hou Ma ◽  
Chin-Hsien Wu ◽  
I-Ming Jou ◽  
Yuan-Kun Tu ◽  
Ching-Hsia Hung ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Dysfunction ◽  
P38 Mapk ◽  
Articular Chondrocytes ◽  
Cartilage Degeneration ◽  
The Elderly ◽  
Human Articular Chondrocytes ◽  
Poly I:C ◽  
Chondrocyte Apoptosis ◽  
Tnf Α

Osteoarthritis (OA) is one of the most common types of arthritis in the elderly people. It has been known that chondrocyte apoptosis occurs in OA cartilage; however, the detailed molecular mechanism remains unclear. In the current study, we aimed to elucidate the role of double-stranded RNA-dependent protein kinase R (PKR) in the TNF-α-caused apoptosis in chondrocytes. Human articular chondrocytes were digested from cartilages of OA subjects who accepted arthroplastic knee surgery. Our results showed that phosphorylation of p38 MAPK was increased after TNF-α stimulation or PKR activation using poly (I:C), and TNF-α-induced p38 MAPK upregulation was inhibited by PKR inhibition, suggesting phosphor-p38 MAPK was regulated by PKR. Moreover, we found that PKR participated in the p53-dependent destruction of AKT following activation of p38 MAPK. The inhibition of AKT led to the reduced expression of PGC-1α, which resulted in mitochondrial dysfunction and increased oxidative stress. We showed that the reduction of oxidative stress using antioxidant Mito TEMPO lowered the TNF-α-induced caspase-3 activation and TUNEL-positive apoptotic cells. The diminished apoptotic response was also observed after repression of PKR/p38 MAPK/p53/AKT/PGC-1α signaling. Taken together, we demonstrated that the aberrant mitochondrial biogenesis and increased oxidative stress in chondrocytes after TNF-α stimulation were mediated by PKR, which may contribute to the chondrocyte apoptosis and cartilage degeneration in OA.

Differentiation regulates interleukin-1β-induced cyclo-oxygenase-2 in human articular chondrocytes: role of p38 mitogen-activated protein kinase

2002 ◽  
Vol 362 (2) ◽  
pp. 367-373 ◽  
Author(s):  
Béatrice THOMAS ◽  
Sylvie THIRION ◽  
Lydie HUMBERT ◽  
Lujian TAN ◽  
Mary B. GOLDRING ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Articular Chondrocytes ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Human Articular Chondrocytes ◽  
Osteoarthritic Cartilage ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Cox 2

Chondrocyte dedifferentiation has been noted in osteoarthritic cartilage, but the contribution of this phenomenon is poorly understood. Interleukin (IL)-1β, the major pro-inflammatory cytokine found in osteoarthritic synovial fluid, induces the dedifferentiation of cultured articular chondrocytes, whereas E-series prostaglandins (PGE) are capable of inducing cell differentiation. Since PGE2 synthesis is up-regulated by IL-1β, we addressed the question of whether the state of chondrocyte differentiation may influence the production of IL-1-induced PGE2 by modulating cyclooxygenase (COX)-2 expression. Immortalized human articular chondrocytes, (tsT/AC62) cultured in monolayer after passage through alginate matrix (alg+) produced 5-fold greater amounts of PGE2 than continuous monolayer cultures (alg-) after stimulation with IL-1β. Moreover, IL-1β induced COX-2 expression at 0.01ng/ml in (alg+) cells, whereas a 100-fold higher dose of cytokine was necessary for stimulation in (alg-) cells. SB203580, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor, completely abolished the IL-1β-induced COX-2 mRNA. Overexpression of p38 MAPK induces a COX-2 reporter, whereas overexpression of dominant negative p38 MAPK represses IL-1β-induced promoter expression. Interestingly, IL-1β-induced p38 MAPK activity was greatly enhanced in (alg+) compared with (alg-) cells. Our results suggest that differentiated articular chondrocytes are highly responsive to IL-1β and that p38 MAPK mediates this response by inducing COX-2 gene expression.

IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-α in human articular chondrocytes in vitro

Cytokine ◽  
2008 ◽  
Vol 44 (3) ◽  
pp. 377-385 ◽  
Author(s):  
R.D. Müller ◽  
T. John ◽  
B. Kohl ◽  
A. Oberholzer ◽  
T. Gust ◽  
...  
Keyword(s):  
Gene Expression ◽  
Articular Chondrocytes ◽  
Cartilage Matrix ◽  
Human Articular Chondrocytes ◽  
Matrix Gene ◽  
Tnf Α

scholarly journals Hypoxia potentiates the BMP-2 driven COL2A1 stimulation in human articular chondrocytes via p38 MAPK

2016 ◽  
Vol 24 (5) ◽  
pp. 856-867 ◽  
Author(s):  
J.E. Lafont ◽  
F.-A. Poujade ◽  
M. Pasdeloup ◽  
P. Neyret ◽  
F. Mallein-Gerin
Keyword(s):  
P38 Mapk ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes

scholarly journals HIV-1 transactivator protein Tat induces proliferation and TGF beta expression in human articular chondrocytes

1994 ◽  
Vol 124 (3) ◽  
pp. 365-371 ◽  
Author(s):  
M Lotz ◽  
I Clark-Lewis ◽  
V Ganu
Keyword(s):  
Growth Factor ◽  
Articular Chondrocytes ◽  
Full Length ◽  
Human Articular Chondrocytes ◽  
Beta Activity ◽  
Mrna Levels ◽  
Tgf Beta ◽  
Chondrocyte Proliferation ◽  
Tat Peptides ◽  
Hiv 1

The human immunodeficiency virus-1 (HIV-1) protein Tat binds to cell surface antigens and can regulate cellular responses. Tat has similar immunosuppressive effects as transforming growth factor-beta (TGF beta) and both inhibit lymphocyte proliferation. TGF beta is expressed by primary human articular chondrocytes and is their most potent growth factor. The present study analyzed the interactions of TGF beta and HIV Tat in the regulation of human articular chondrocytes. Synthetic or recombinant full-length Tat (1-86) induced chondrocyte proliferation and this was of similar magnitude as the response to TGF beta. Tat peptides that did not contain the RGD motif had similar chondrocyte stimulatory activity as full-length Tat. Among a series of Tat peptides, peptide 38-62 which contains the basic domain was the only one active, suggesting that this region is responsible for the effects on chondrocyte proliferation. Full-length Tat and peptide 38-62 synergized with TGF beta and induced proliferative responses that were greater than those obtained with any combination of the known chondrocyte growth factors. Further characterization of the interactions between Tat and TGF beta showed that Tat increased synthesis and TGF beta activity and TGF beta 1 mRNA levels. The stimulatory effects of Tat and peptide 38-62 on chondrocyte proliferation were reduced by neutralizing antibodies to TGF beta and by TGF beta antisense oligonucleotides. These results identify a virally encoded protein and a synthetic peptide derived from it as novel and potent chondrocyte growth stimuli which act at least in part through the induction of TGF beta.

TGFβ inhibits LPS-induced chemokine mRNA stabilization

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1178-1185 ◽  
Author(s):  
Yalei Dai ◽  
Shyamsree Datta ◽  
Michael Novotny ◽  
Thomas A. Hamilton
Keyword(s):  
Gene Expression ◽  
P38 Mapk ◽  
Half Life ◽  
Transforming Growth Factor ◽  
Luciferase Expression ◽  
Mrna Levels ◽  
Chemokine Gene ◽  
Mrna Stabilization ◽  
Chemokine Gene Expression ◽  
In Cells

Abstract The mechanisms involved in anti-inflammatory action of transforming growth factor β (TGFβ) have been examined by evaluating its effect on chemokine gene expression in mouse macrophages. Lipopolysaccharide (LPS)–stimulated expression of the CXC chemokines KC and MIP-2 was selectively reduced by TGFβ in a time- and protein synthesis–dependent process. While TGFβ had a modest effect on transcription of the KC and MIP-2 mRNAs as measured by nuclear run-on, it had no effect on LPS-stimulated luciferase expression driven by the KC promoter nor on the activation of nuclear factor κB (NFκB) DNA-binding activity and transactivation function. Interestingly, KC mRNA levels were markedly reduced by TGFβ treatment in cells transfected with KC genomic or cDNA constructs driven from either the KC or cytomegalovirus (CMV) promoters, demonstrating the importance of sequences within the mature mRNA and suggesting that suppression may involve a posttranscriptional mechanism. In support of this possibility, LPS stimulation prolonged the half-life of KC mRNA and this stabilization response was blocked in cells treated with TGFβ. Examination of KC mRNA expressed under control of a tetracycline-responsive promoter demonstrated that TGFβ prevented stabilization of KC mRNA, in response to LPS but did not alter KC mRNA half-life directly. KC mRNA stabilization by LPS was dependent on activation of p38 mitogen-activated protein kinase (MAPK) activity, and TGFβ treatment inhibited p38 MAPK activation. These findings support the hypothesis that TGFβ-mediated suppression of chemokine gene expression involves antagonism of LPS-stimulated KC mRNA stabilization via inhibition of p38 MAPK.

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