Viktor Wixler

The LIM-only protein FHL2 interacts with β-catenin and promotes differentiation of mouse myoblasts

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified β-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of β-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts β-catenin–mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell–specific manner. After injection into Xenopus embryos, FHL2 inhibited the β-catenin–induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with β-catenin.

3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity.

The regulatory subunit of protein kinase CK2 is a specific A‐Raf activator

Two protein kinases that are involved in proliferation and oncogenesis but so far were thought to be functionally independent are Raf and CK2. The Raf signaling pathway is known to play a critical role in such fundamental biological processes as cellular proliferation and differentiation. Abnormal activation of this pathway is potentially oncogenic. Protein kinase CK2 exhibits enhanced levels in solid human tumors and proliferating tissue. In a two‐hybrid screen of a mouse‐embryo cDNA library we detected an interaction between A‐Raf and CK2β subunit. This binding was specific, as no interaction between CK2β and B‐Raf or c‐Raf‐1 was observed. Regions critical for this interaction were localized between residues 550 and 569 in the A‐Raf kinase domain. A‐Raf kinase activity was enhanced 10‐fold upon coexpression with CK2β in Sf9 cells. The α subunit of CK2 abolishes this effect. This is the first demonstration of both a direct Raf‐isoform‐specific activation and a regulatory role for CK2β independent of the CK2α subunit. The present data thus link two different protein kinases that were thought to work separately in the cell.© 1997 Federation of European Biochemical Societies.

Deficiency in the LIM-only protein Fhl2 impairs skin wound healing

After skin wounding, the repair process is initiated by the release of growth factors, cytokines, and bioactive lipids from injured vessels and coagulated platelets. These signal molecules induce synthesis and deposition of a provisional extracellular matrix, as well as fibroblast invasion into and contraction of the wounded area. We previously showed that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of the LIM-only protein Fhl2 in response to activation of the RhoA GTPase (Muller, J.M., U. Isele, E. Metzger, A. Rempel, M. Moser, A. Pscherer, T. Breyer, C. Holubarsch, R. Buettner, and R. Schule. 2000. EMBO J. 19:359–369; Muller, J.M., E. Metzger, H. Greschik, A.K. Bosserhoff, L. Mercep, R. Buettner, and R. Schule. 2002. EMBO J. 21:736–748.). We demonstrate impaired cutaneous wound healing in Fhl2-deficient mice rescued by transgenic expression of Fhl2. Furthermore, collagen contraction and cell migration are severely impaired in Fhl2-deficient cells. Consequently, we show that the expression of α-smooth muscle actin, which is regulated by Fhl2, is reduced and delayed in wounds of Fhl2-deficient mice and that the expression of p130Cas, which is essential for cell migration, is reduced in Fhl2-deficient cells. In summary, our data demonstrate a function of Fhl2 as a lipid-triggered signaling molecule in mesenchymal cells regulating their migration and contraction during cutaneous wound healing.

The influenza virus PB1-F2 protein has interferon antagonistic activity.

Abstract PB1-F2 is a nonstructural protein of influenza viruses encoded by the PB1 gene segment from a +1 open reading frame. It has been shown that PB1-F2 contributes to viral pathogenicity, although the underlying mechanisms are still unclear. Induction of type I interferon (IFN) and the innate immune response are the first line of defense against viral infection. Here we show that influenza A viruses (IAVs) lacking the PB1-F2 protein induce an enhanced expression of IFN-β and IFN-stimulated genes in infected epithelial cells. Studying molecular mechanisms underlying the PB1-F2-mediated IFN antagonistic activity showed that PB1-F2 interferes with the RIG-I/MAVS protein complex thereby inhibiting the activation of the downstream transcription factor IFN regulatory factor 3. These findings were also reflected in in vivo studies demonstrating that infection with PR8 wild-type (wt) virus resulted in higher lung titers and a more severe onset of disease compared with infection with its PB1-F2-deficient counterpart. Accordingly, a much more pronounced infiltration of lungs with immune cells was detected in mice infected with the PB1-F2 wt virus. In summary, we demonstrate that the PB1-F2 protein of IAVs exhibits a type I IFN-antagonistic function by interfering with the RIG-I/MAVS complex, which contributes to an enhanced pathogenicity in vivo.

Differential regulation of Raf isozymes by growth versus differentiation inducing factors in PC12 pheochromocytoma cells.

PC12 pheochromocytoma cells possess four known MEK activators: A‐, B‐, c‐Raf‐1 and MEKK. In order to examine whether differentiation factors or growth factors have a Raf isozyme preference for activation of the mitogenic cytoplasmic Raf‐MEK‐MAPK protein kinase cascade, the activation kinetics of these enzymes in response to epidermal growth factor (EGF) and nerve growth factor (NGF) were compared. An initial activation of all three Raf kinases was noticed, but only A‐ and B‐Raf showed sustained activation by NGF, which was not seen after EGF treatment. Furthermore, expression of oncogenic versions of all three Raf kinases as well as a potentially Raf‐independent MEK activator, v‐Mos, leads to activation of MAPK and to differentiation of PC12 cells. These data suggest a differential regulation of Raf kinases and that probably no alternative Raf substrates are involved in differentiation processes of PC12 cells.

FHL2 regulates cell cycle-dependent and doxorubicin-induced p21Cip1/Waf1 expression in breast cancer cells.

The transcriptional cofactor FHL2 interacts with a broad variety of transcription factors and its expression is often deregulated in various types of cancer. Here we analyzed for the first time the molecular function of FHL2 in breast cancer. FHL2 is overexpressed in almost all human mammary carcinoma samples tested but not in normal breast tissues and only low levels of FHL2 expression were present in four premalignant ductal carcinoma in situ (DCIS). Cell cycle analysis revealed an upregulation of endogenous FHL2 towards G2/M in MDA-MB 231 cells and an accelerated G2/M transition when FHL2 expression was suppressed in these cells. In search for G2/M specific target genes regulated by FHL2, we found that expression of the cell cycle inhibitor p21Cip1/Waf1 (hereafter p21) is dependent on FHL2 in MDA-MB 231 breast cancer cells. Downregulation of FHL2 by shRNA abrogated the cell cycle dependent upregulation of p21 as well as the induction of p21 in response to treatment with the DNA damaging agent doxorubicin. FHL2-dependent p21 expression occurs in a p53-independent manner and p21 expression can be downregulated by specific inhibition of mitogen-activated protein kinases (MAPKs), implicating an involvement of MAPK signaling in this regulation. Analysis of FHL2 contribution to the MAPK signaling identified FHL2 as an important downstream effector of MAPKs in breast cancer cells, capable of transactivating endogenous AP1 target genes as well as AP1 dependent reporter genes. Finally, downregulation of FHL2 reduces the ability of MDA-MB 231 cells to form colonies in soft agar, while FHL2 overexpression enhances colony formation of breast cancer cells. Thus, our findings indicate that overexpression of the transcriptional cofactor FHL2 contributes to breast cancer development by mediating transcriptional activation of MAPK target genes known to be involved in cancer progression, such as p21.

A new splice variant of the human guanylate-binding protein 3 mediates anti-influenza activity through inhibition of viral transcription and replication

Guanylate‐binding proteins (GBPs) belong to the family of large GTPases that are induced in response to interferons. GBPs contain an N‐terminal globular GTPase domain and a C‐terminal α‐helical regulatory domain that are connected by a short middle domain. Antiviral activity against vesicular stomatitis virus and encephalomyocarditis virus has been shown for hGBP‐1; however, no anti‐influenza virus properties for GBPs have been described to date. Here we show that hGBP‐1 and hGBP‐3 possess anti‐influenza viral activity. Furthermore, we have identified a novel splice variant of hGBP‐3, named hGBP‐3ΔC, with a largely modified C‐terminal α‐helical domain. While all three GBP isoforms were up‐regulated on influenza virus infection, hGBP‐3ΔC showed the most prominent antiviral activity in epithelial cells. Mutational analysis of hGBPs revealed that the globular domain is the principal antiviral effector domain, and GTP‐binding, but not hydrolysis, is necessary for antiviral action. Furthermore, we showed that hGBP‐3ΔC strongly represses the activity of the viral polymerase complex, which results in decreased synthesis of viral vRNA, cRNA, mRNA, and viral proteins, as well.—Nordmann, A., Wixler, L., Boergeling, Y., Wixler, V., Ludwig, S. A new splice variant of the human guanylate‐binding protein 3 mediates anti‐influenza activity through inhibition of viral transcription and replication. FASEB J. 26, 1290‐1300 (2012). www.fasebj.org

Phosphatidylinositol‐3‐kinase (PI3K) is activated by influenza virus vRNA via the pathogen pattern receptor Rig‐I to promote efficient type I interferon production

The phosphatidylinositol‐3‐kinase (PI3K) was identified to be activated upon influenza A virus (IAV) infection. An early and transient induction of PI3K signalling is caused by viral attachment to cells and promotes virus entry. In later phases of infection the kinase is activated by the viral NS1 protein to prevent premature apoptosis. Besides these virus supporting functions, it was suggested that PI3K signalling is involved in dsRNA and IAV induced antiviral responses by enhancing the activity of interferon regulatory factor‐3 (IRF‐3). However, molecular mechanisms of activation remained obscure. Here we show that accumulation of vRNA in cells infected with influenza A or B viruses results in PI3K activation. Furthermore, expression of the RNA receptors Rig‐I and MDA5 was increased upon stimulation with virion extracted vRNA or IAV infection. Using siRNA approaches, Rig‐I was identified as pathogen receptor necessary for influenza virus vRNA sensing and subsequent PI3K activation in a TRIM25 and MAVS signalling dependent manner. Rig‐I induced PI3K signalling was further shown to be essential for complete IRF‐3 activation and consequently induction of the type I interferon response. These data identify PI3K as factor that is activated as part of the Rig‐I mediated anti‐pathogen response to enhance expression of type I interferons.

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

We have described the scaffolding protein FHL2 as a component of focal adhesion structures, to which it is recruited via binding to both αa‐ or β‐integrin subunits. Using mesenchymal stem cells from wild‐type and FHL2‐knockout mice, we show here that inactivation of FHL2 leads to impaired assembly of extracellular matrix proteins on the cell surface and to impaired bundling of focal adhesions. Both altered properties can be restored by reexpression of recombinant FHL2 protein in FHL2‐null cells. Molecular analysis of integrin‐mediated signaling revealed a higher phosphorylation of FAK at tyrosine 925 in FHL2‐knockout cells compared to their wild‐type counterpart. Consequently, the activation of the mitogenic kinase ERK was more pronounced in knockout cells on cell adhesion. The growth factor‐induced activation of ERK, however, was not altered. The perturbed organization of extracellular matrix on FHL2‐null cells was improved when the increased activation of MAPK was inhibited. Our findings point to a role of FHL2 in bundling of focal adhesion structures, in integrin‐mediated ERK activation, and subsequently in proper allocation of matrix proteins on the cell surface.—Park, J., Will, C., Martin, B., Gullotti, L., Friedrichs, N., Buettner, R., Schneider, H., Ludwig, S., Wixler, V. Deficiency in the LIM‐only protein FHL2 impairs assembly of extracellular matrix proteins. FASEB J. 22, 2508–2520 (2008)