Higher TRIP-1 level explains diminished collagen contraction ability of fetal versus adult fibroblasts

Navarro, Angels; Rezaiekhaligh, Mo; Keightley, J. Andrew; Mabry, Sherry M; Perez, Ricardo E.; Ekekezie, Ikechukwu I

doi:10.1152/ajplung.00012.2009

Cited by 12 publications

(11 citation statements)

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“…We previously found that fetal human lung fibroblasts (HLF-F) lesser ability for collagen contraction is due to higher TRIP-1 protein expression [22]. To assess whether TRIP-1 drives fibroblast acquisition of myofibroblast phenotype and function in human lung fibroblasts, TRIP-1 siRNA-transfected versus control siRNA-transfected HLF-F were embedded in a 3D collagen matrix and studied for collagen contraction response and α-SMA expression.…”

Section: Resultsmentioning

confidence: 99%

“…Fibroblasts were trypsinized, resuspended in cold, serum-free DMEM, and added to the collagen mixture at a final concentration of 5 × 10 5 cells/ml with 0.75 mg/ml as final collagen concentration [22]. Aliquots (in triplicate) were cast onto 24-well plates and allowed to solidify at 37°C (30–45 minutes), after which serum-free media was added, incubated, periodically observed for contraction, and photographed.…”

Section: Methodsmentioning

confidence: 99%

“…Total cell lysates were prepared (unless specified in figure legend), and proteins were separated by SDS-PAGE, transferred to PVDF membranes and detected with ECL from GE Healthcare Biosciences (Piscataway, NJ) as previously described [22], using polyclonal rabbit anti-TRIP-1 from Abcam (1:1000), mouse monoclonal antibodies against smooth muscle actin (1:1000) and tubulin (1:10000) from Sigma-Aldrich and P-Ser473-Akt (1:1000), Smad3 (rabbit monoclonal C67H9, 1:1000), Cleaved Caspase-3 (rabbit monoclonal D175, clone 5A1E, 1:1000 using nitrocellulose 0.2 μm pore size instead of PVDF membranes) from Cell Signaling Technologies, and anti-actin (polyclonal goat I-19, 1:1000) from Santa Cruz Biotechnologies (Santa Cruz, CA). Western-blots were developed using ECL (chemiluminescence), and densitometric scans were quantified using Alpha-Innotech and AlphaEaseFC Imaging Software (Protein Simple, Santa Clara, CA).…”

Section: Methodsmentioning

confidence: 99%

“…More recently they showed that TRIP-1 is secreted in the provisional bone matrix and is not only present in the cytosol, but also in the nucleus of osteoblasts [21]. We have reported that TRIP-1 is a negative regulator of fibroblast collagen contraction and modulates epithelial-mesenchymal transition (EMT) of lung epithelial cells, which would suggest it has the potential to influence execution of fibrotic pathology [22-24]. Myofibroblasts are the critical effector cells in the pathogenesis of fibrotic remodeling, but whether or not TRIP-1 modulates fibroblast trans-differentiation to a myofibroblast phenotype has not been explored.…”

Section: Introductionmentioning

confidence: 99%

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TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation

et al. 2014

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BackgroundMyofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known.MethodsTRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis.ResultsThe down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis.ConclusionsTRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation

et al. 2014

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“…We showed recently (20) that TRIP-1 is a novel modulator of fibroblast collagen contraction, an important step in wound repair and an event stimulated by TGF-␤1 action.…”

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TRIP-1 regulates TGF-β1-induced epithelial-mesenchymal transition of human lung epithelial cell line A549

Perez

Navarro

Rezaiekhaligh

et al. 2011

American Journal of Physiology-Lung Cellular and Molecular Phys

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Perez RE, Navarro A, Rezaiekhaligh MH, Mabry SM, Ekekezie II. TRIP-1 regulates TGF-␤1-induced epithelial-mesenchymal transition of human lung epithelial cell line A549. Am J Physiol Lung Cell Mol Physiol 300: L799 -L807, 2011. First published March 4, 2011 doi:10.1152/ajplung.00350.2010.-Epithelialmesenchymal transition (EMT) is a process by which epithelial cells undergo conversion to a mesenchymal phenotype contributing to wound repair by fibrosis and to cancer cell acquisition of invasive ability. Recently, we showed that type II TGF-␤ receptor interacting protein-1 (TRIP-1), a protein identified as a phosphorylation target of the TGF-␤ type II receptor kinase and as a functional component of eukaryotic translation initiator factor 3 (eiF3) multiprotein complex, is a novel modulator of fibroblast collagen contraction, an important step in wound repair stimulated by TGF-␤1 action. TGF-␤1 drives EMT, but it is not known whether TRIP-1 expression influences EMT induction. To investigate whether TRIP-1 plays a role in EMT induction we studied the effect of downregulating TRIP-1 expression in the well-characterized A549 model of TGF-␤1 induction of EMT. Here we report that short hairpin RNA (shRNA)-mediated depletion of TRIP-1 gene transcripts in A549 cells promotes EMT as assessed by changes in phenotypic markers, morphology, and migrative ability. Knockdown of TRIP-1 dramatically increased A549 responsiveness to TGF-␤1 induction of EMT. Mechanistically, a pathway involving increased TGF-␤ type II receptor level, enhanced Smad3 phosphorylation, and the transcription factor SLUG is implicated. Altogether, the findings point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells.transforming growth factor-␤; type II transforming growth factor-␤ receptor interacting protein-1

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Semaphorin 4A enhances lung fibrosis through activation of Akt via PlexinD1 receptor

et al. 2015

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Semaphorin 4A plays a regulatory role in immune function and angiogenesis. However, its specific involvement in controlling lung fibrosis, a process that is closely related to angiogenesis and inflammation is still poorly understood. In the present study, we show that treatment of Sema4A on normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including alpha-smooth muscle actin (alpha-SMA), ezrin, moesin, and paxillin. We confirm that Sema4A enhances the ability of lung fibroblasts to contract collagen gel. Sema4A treatment led to resistance to apoptosis in normal lung fibroblasts. Relative to normal lung fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease Systemic Sclerosis (SSc) showed elevated Sema4A secretion, enhanced alpha-SMA, ezrin, moesin, and paxillin expression, and high ability to induce collagen gel contraction. Using neutralizing antibody against Sema4A receptor, PlexinD1, we found that endogenous Sema4A signalling in SSc fibroblast was through PlexinD1 receptor. We then identified the signalling mechanism through which Sema4A-PlexinD1 promotes the ability of normal fibroblasts to contract a collagen gel matrix. Western blot analysis showed that Sema4A activated the Akt pathway in lung fibroblasts, and the specific inhibitor of Akt pathway, Akt inhibitor III, blocked the ability of Sema4A to promote the ability of lung fibroblasts to contract a collagen gel matrix. Thus, blocking Sema4APlexinD1- Akt cascades might be beneficial in reducing pulmonary fibrosis.

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Higher TRIP-1 level explains diminished collagen contraction ability of fetal versus adult fibroblasts

Cited by 12 publications

References 33 publications

TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation

TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation

TRIP-1 regulates TGF-β1-induced epithelial-mesenchymal transition of human lung epithelial cell line A549

Semaphorin 4A enhances lung fibrosis through activation of Akt via PlexinD1 receptor

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