Deficiency in the LIM‐only protein FHL2 impairs assembly of extracellular matrix proteins

Park, Jung; Will, Carola; Martin, Bernd; Gullotti, Lucia; Friedrichs, Nicolaus; Buettner, Reinhard; Schneider, Holm; Ludwig, Stephan; Wixler, Viktor

doi:10.1096/fj.07-095521

Cited by 38 publications

(52 citation statements)

References 53 publications

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“…These findings are consistent with previous report showing that inactivation of FHL2 leads to impaired assembly of extracellular matrix proteins on the surface and to impaired bundling of focal adhesions. 22 Furthermore, previous report showed an essential role of CD18 and its ligand ICAM-1 in mediating EPC recruitment and the subsequent functional effects on the infarcted tissues. 23 Using in vitro assays, we have for the first time demonstrated the detrimental effect of FHL2 siRNA on the migration and vasculogenesis activity of EPCs, which implies a multifunctional role for FHL2 in ischemiainduced neovascularization.…”

Section: Fhl2mentioning

confidence: 97%

Deletion of FHL2 Gene Impaired Ischemia-Induced Blood Flow Recovery by Modulating Circulating Proangiogenic Cells

Huang

Chen

Lin

et al. 2013

ATVB

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Objective-The four and a half Lin11, Isl-1 and Mec-3 (LIM) domain protein 2 (FHL2) is a member of the four and a half LIM domain-only (FHL) gene family, and has been shown to play an important role in inhibiting inflammatory angiogenesis. Here, we tested the hypothesis that impaired ischemia-induced neovascularization in mice lacking FHL2 is related to a defect in proangiogenic cell mobilization and functions in vasculogenesis. Approach and Results-Unilateral hindlimb ischemia surgery was conducted in FHL2 −/− mice and wild-type (FHL2 +/+ ) mice. After hindlimb ischemia surgery, expression of FHL2 protein was noted in ischemic tissues of wild-type mice. All FHL2-null mice (100%) suffered from spontaneous foot amputation, but only 20% of wild-type mice had ischemiainduced foot amputation after ischemic surgery. Blood flow recovery was significantly impaired in FHL2 −/− mice when compared with that in wild-type mice as determined by laser Doppler imaging. Histological analysis revealed that the capillary density in the ischemic limb was increased in wild-type mice, whereas no such increase was noted in FHL2 −/− mice. Flow cytometry demonstrated that the number of CD34 + or CD34 + /Sca-1 + /Flk-1 + in peripheral blood after ischemic surgery significantly decreased in FHL2-null mice than those in wild-type mice after hindlimb ischemia surgery. FHL2 deficiency impaired ex vivo angiogenesis in mouse aortic-ring culture assay, which revealed that the mean density of the microvessels was significantly higher in the wild-type aorta than in the FHL2 −/− aorta. Western blot analysis showed that vascular endothelial growth factor (VEGF), interleukin-6, matrix metalloproteinase-2, matrix metalloproteinase-9, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 levels were significantly downregulated in ischemic muscles in FHL2-null mice compared with wild-type mice. Deletion of FHL2 protein by FHL2 small interfering RNA impaired VEGF production under hypoxia conditions, and also suppressed endothelial progenitor cell angiogenic functions, but these effects could be recovered by administration of VEGF. Conclusions-Deficiency of FHL2 impairs ischemia-induced neovascularization, and these suppressive effects may occur through a reduction in proangiogenic cell mobilization, migration, and vasculogenesis functions. The four and a half Lin11, Isl-1 and Mec-3 (LIM) domain (FHL) protein family is a newly identified group of proteins containing FHLs. This family consists of 6 members-FHL1, FHL2, FHL3, FHL4, FHL5, and activator of cAMP-responsive element modulator in testes. [11][12][13] Recent studies have indicated that human four and a half LIM-only protein family members, including FHL2, are expressed in a cell-and tissuespecific manner and participate in various cellular processes, such as regulation of gene expression, cytoarchitecture, cell adhesion, cell survival, cell mobility, transcription, and signal transduction.13,14 Increasing evidence shows that FHL2 is abundantly expressed in the vascular system, i...

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Section: Fhl2mentioning

confidence: 97%

Deletion of FHL2 Gene Impaired Ischemia-Induced Blood Flow Recovery by Modulating Circulating Proangiogenic Cells

Huang

Chen

Lin

et al. 2013

ATVB

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“…FHL2 is involved in many processes, including cell cycle regulation (31,41), apoptosis (55,60), differentiation (21,32,40,63), extracellular matrix assembly (48), bone formation (20), and wound healing (28,65). Although the highest expression of FHL2 occurs in the heart, knockout mice are viable and display no overt cardiac phenotype under basal conditions (5,30).…”

mentioning

confidence: 99%

FHL2 Binds Calcineurin and Represses Pathological Cardiac Growth

Hojayev

Rothermel²,

Gillette³

et al. 2012

Molecular and Cellular Biology

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Stress-induced hypertrophic growth of the heart predisposes the heart to arrhythmia, contractile dysfunction, and clinical heart failure. FHL2 (four-and-a-half LIM domain protein 2) is expressed predominantly in the heart, and inactivation of the gene coding for FHL2 leads to exaggerated responsiveness to adrenergic stress. Activation of calcineurin occurs downstream of ␤-adrenergic signaling and is required for isoproterenol-induced myocardial hypertrophy. Based on these facts, we hypothesized that FHL2 suppresses stress-induced activation of calcineurin. FHL2 is upregulated in mouse hearts exposed to isoproterenol, a ␤-adrenergic agonist, and isoproterenol-induced increases in the NFAT target genes RCAN1.4 and BNP were amplified significantly in FHL2 knockout (FHL2 ؊/؊ ) mice compared with levels in wild-type (WT) mice. To determine whether the effect of FHL2 on NFAT target gene transcript levels occurred at the level of transcription, HEK 293 cells and neonatal rat ventricular myocytes (NRVMs) were transfected with a luciferase reporter construct harboring the NFAT-dependent promoters of either RCAN1 or interleukin 2 (IL-2). Consistent with the in vivo data, small interfering RNA (siRNA) knockdown of FHL2 led to increased activation of these promoters by constitutively active calcineurin or the calcium ionophore ionomycin. Importantly, activation of the RCAN1 promoter by ionomycin, in control and FHL2 knockdown cells, was abolished by the calcineurin inhibitor cyclosporine, confirming the calcineurin dependence of the response. Overexpression of FHL2 inhibited activation of both NFAT reporter constructs. Furthermore, NRVMs overexpressing FHL2 exhibited reduced hypertrophic growth in response to constitutively active calcineurin, as measured by cell cross-sectional area and fetal gene expression. Finally, immunostaining in isolated adult cardiomyocytes revealed colocalization of FHL2 and calcineurin predominantly at the sarcomere and activation of calcineurin by endothelin-1-facilitated interaction between FHL2 and calcineurin. FHL2 is an endogenous, agonist-dependent suppressor of calcineurin. E pidemiological evidence links left ventricular (LV) hypertrophy with adverse cardiovascular events, including heart failure and death (1,13,35,36). Consistent with this, therapies that improve clinical outcomes are often associated with regression of ventricular hypertrophy (11,19,46). However, whereas significant strides have been made in elucidating the molecular circuitry governing pathological cardiac remodeling (23), few therapies in clinical use target cell growth mechanisms directly.Hypertrophic growth of the heart in response to a variety of pathological stresses is an initially adaptive response that, left unchecked, often progresses to heart failure (25). In many instances, the intracellular protein phosphatase calcineurin is a major mediator of stress-induced cardiac hypertrophy. Upon activation, calcineurin dephosphorylates NFAT (nuclear factor of activated T cells), which, in turn, translocates into the n...

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“…9 Furthermore, FHL2 is critically involved in matrix assembly allowing migration of cells into the wound area. 10 Analogous to wound healing invasive carcinomas generate a specialised tumour stroma and deposit a matrix facilitating invasion into the surrounding tissue. 11 This tissue remodelling is induced by pro-metastatic factors, such as transforming growth factor (TGF)-b1, which is secreted by the tumour cells inducing the conversion of mesenchymal cells into myofibroblasts.…”

mentioning

confidence: 99%

FHL2 expression in peritumoural fibroblasts correlates with lymphatic metastasis in sporadic but not in HNPCC-associated colon cancer

Gullotti

Czerwitzki

Kirfel

et al. 2011

Laboratory Investigation

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Four and a half LIM domain protein-2 (FHL2) is a component of the focal adhesion structures and has been suggested to have an important role in cancer progression. This study analyses the role of FHL2 in peritumoural fibroblasts of sporadic and hereditary non-polyposis colorectal cancer (HNPCC). Tissue specimens of 48 sporadic and 49 hereditary colon cancers, respectively, were stained immunohistochemically for FHL2, transforming growth factor (TGF)-b1 ligand and a-SMA. Myofibroblasts at the tumour invasion front co-expressed a-SMA and FHL2. Sporadic colon cancer but not HNPCC cases showed a correlation between TGF-b1 expression of the invading tumour cells and FHL2 staining of peritumoural myofibroblasts. Overexpression of FHL2 in peritumoural myofibroblasts correlated to lymphatic metastasis in sporadic colon cancer but not in HNPCC. In cultured mouse fibroblasts, TGF-b1 treatment induced myofibroblast differentiation, stimulated FHL2 protein expression and elevated number of migratory cells in transwell motility assays, suggesting that FHL2 is regulated downstream of TGF-b. Physical contact of colon cancer cells and myofibroblasts via FHL2-positive focal adhesions was detected in human colon carcinoma tissue and in co-culture assays using sporadic as well as HNPCC-derived tumour cell lines. Our data provide strong evidence for an important role of FHL2 in the progression of colon cancers. Tumour-secreted TGF-b1 stimulates FHL2 protein expression in peritumoural fibroblasts, probably facilitating the invasion of tumour glands into the surrounding tissue by enhanced myofibroblast migration and tight connection of fibroblasts to tumour cells via focal adhesions. These findings are absent in HNPCC-associated colon cancers in vivo and may contribute to a less invasive and more protruding tumour margin of microsatellite instable carcinomas. Four and a half LIM domain protein-2 (FHL2) was first identified as a protein differentially expressed in human myoblasts and rhabdomyosarcoma cells, and thus named DRAL (downregulated in rhabdomyosarcoma LIM protein 1 ). FHL2 is a LIM-only protein with four complete and one N-terminal half LIM domains that mediate protein-protein interactions. 2 FHL2 associates with integrin receptors to form focal adhesion contacts and binds signal transducers such as b-catenin. 3-5 Triggered by lipid-induced signalling, such as sphingosine-1-phosphate, FHL2 translocates into the nucleus where it binds several transcription factors including serum response factor, AP1 and androgen receptor and functions as a coactivator or a corepressor to modulate gene expression. [6][7][8] We showed previously that FHL2 is strongly upregulated in mesenchymal cells of wounded skin, and demonstrated a function of FHL2 in myofibroblasts regulating their migration and contraction during cutaneous wound healing. 9 Furthermore, FHL2 is critically involved in matrix assembly allowing migration of cells into the wound area. 10 Analogous to wound healing invasive carcinomas generate a specialised tumour stroma and de...

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Deficiency in the LIM‐only protein FHL2 impairs assembly of extracellular matrix proteins

Cited by 38 publications

References 53 publications

Deletion of FHL2 Gene Impaired Ischemia-Induced Blood Flow Recovery by Modulating Circulating Proangiogenic Cells

Deletion of FHL2 Gene Impaired Ischemia-Induced Blood Flow Recovery by Modulating Circulating Proangiogenic Cells

FHL2 Binds Calcineurin and Represses Pathological Cardiac Growth

FHL2 expression in peritumoural fibroblasts correlates with lymphatic metastasis in sporadic but not in HNPCC-associated colon cancer

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