Crystal structure of human transgelin

Transgelin (TAGLN), also known as smooth muscle protein 22 (SM22), is a highly conserved protein found in smooth muscle tissues of adult vertebrates. Abolition of transgelin gene expression by the oncogenic Ras may be an important early event in tumor progression and a diagnostic marker for breast and colon cancer development. Transgelin contains a single calponin homology (CH) domain. However, the question of whether this single CH domain can bind actin remains open. Here we report the 2.3 A resolution crystal structure of full length human transgelin, whose main structural feature is confirmed to be a CH domain. Secondary structures of CH domains from different proteins were analyzed and conserved residues were identified that maintain similar tertiary structures.     

1st National Congress of the Italian Proteome Society

Transgelin is an abundant protein of smooth muscle cells, where its role has been primarily studied. As a protein affecting dynamics of the actin cytoskeleton via stabilization of actin filaments, transgelin is both directly and indirectly involved in many cancer-related processes such as migration, proliferation, differentiation or apoptosis. Transgelin was previously reviewed as a tumor suppressor; however, recent data based on a number of proteomics studies indicate its pro-tumorigenic role, for example, in colorectal or hepatocellular cancer. We summarize these contradictory observations in both clinical and functional proteomics projects and analyze the role of transgelin in tumors in detail. Generally, the expression and biological role of transgelin seem to differ among various types of tumor cells and stroma, and possibly change during tumor progression. We also overview the recent data on transgelin-2, a sequence homolog of transgelin, whose role in the tumor development might be contradictory to the role of transgelin.     

Expression cloning identifies transgelin (SM22) as a novel repressor of 92-kDa type IV collagenase (MMP-9) expression

The 92-kDa gelatinase (MMP-9) expression is prerequisite for tissue remodeling in physiology and cancer. However, there are few known regulators of MMP-9 expression. Using an expression cloning strategy, we identified transgelin (SM22), a 22-25-kDa actin-binding protein localized to the cell membrane and cytoplasm, as a novel regulator of MMP-9 expression. Overexpression of a SM22 cDNA in HT1080 cells decreased MMP-9 mRNA/protein levels and diminished in vitro invasion of the latter rescued with exogenous MMP-9. Conversely, small interfering RNA-mediated knockdown of SM22 elevated MMP-9 synthesis, and uterus from SM22-null mice showed strong MMP-9 immunoreactivity compared with wild type animals. The ability of SM22 to repress MMP-9 expression required an intact amino terminus calponin homology domain. MMP-9 expression is driven by ERK signaling and SM22 targeted this pathway as evidenced by (a) the transience in MAPK activation and (b) blunted stimulation of the MMP-9 promoter by a constitutively active MEK expression vector. Progressive deletion analysis located the SM22 responsive region of the MMP-9 promoter to the proximal 90-bp region harboring an AP-1 motif subsequently implicated by site-directed mutagenesis. Furthermore, nuclear extract from the SM22 transfectants showed diminished c-Fos binding to this motif and SM22 expression reduced the activity of an AP-1-driven reporter by 75%. Thus, SM22 adds to a short list of repressors of MMP-9 expression, achieving this by reducing AP-1-dependent trans-activation of the gene by way of compromised ERK activation. Diminished transgelin expression in several cancers may thus partly account for the elevated MMP-9 expression evident in these tumors.     

Transgelin Silencing Induces Different Processes in Different Breast Cancer Cell Lines

Transgelin is a protein reported to be a marker of several cancers. However, previous studies have shown both up‐ and down‐regulation of transgelin in tumors when compared with non‐tumor tissues and the mechanisms whereby transgelin may affect the development of cancer remain largely unknown. Transgelin is especially abundant in smooth muscle cells and is associated with actin stress fibers. These contractile structures participate in cell motility, adhesion, and the maintenance of cell morphology. Here, the role of transgelin in breast cancer is focused on. Initially, the effects of transgelin on cell migration of the breast cancer cell lines, BT 549 and PMC 42, is studied. Interestingly, transgelin silencing increased the migration of PMC 42 cells, but decreased the migration of BT 549 cells. To clarify these contradictory results, the changes in protein abundances after transgelin silencing in these two cell lines are analyzed using quantitative proteomics. The results confirmed the role of transgelin in the migration of BT 549 cells and suggest the involvement of transgelin in apoptosis and small molecule biochemistry in PMC 42 cells. The context‐dependent function of transgelin reflects the different molecular backgrounds of these cell lines, which differ in karyotypes, mutation statuses, and proteome profiles.     

Nerve growth factor activation of Erk-1 and Erk-2 induces matrix metalloproteinase-9 expression in vascular smooth muscle cells.

In response to vascular injury, smooth muscle cells migrate from the media into the intima, where they contribute to the development of neointimal lesions. Increased matrix metalloproteinase (MMP) expression contributes to the migratory response of smooth muscle cells by releasing them from their surrounding extracellular matrix. MMPs may also participate in the remodeling of extracellular matrix in vascular lesions that could lead to plaque weakening and subsequent rupture. Neurotrophins and their receptors, the Trk family of receptor tyrosine kinases, are expressed in neointimal lesions, where they induce smooth muscle cell migration. We now report that nerve growth factor (NGF)-induced activation of the TrkA receptor tyrosine kinase induces MMP-9 expression in both primary cultured rat aortic smooth muscle cells and in a smooth muscle cell line genetically manipulated to express TrkA. The response to NGF was specific for MMP-9 expression, as the expression of MMP-2, MMP-3, or the tissue inhibitor of metalloproteinase-2 was not changed. Activation of the Shc/mitogen-activated protein kinase pathway mediates the induction of MMP-9 in response to NGF, as this response is abrogated in cells expressing a mutant TrkA receptor that does not bind Shc and by pretreatment of cells with the MEK-1 inhibitor, U0126. Thus, these results indicate that the neurotrophin/Trk receptor system, by virtue of its potent chemotactic activity for smooth muscle cells and its ability to induce MMP-9 expression, is a critical mediator in the remodeling that occurs in the vascular wall in response to injury.     

Transgelin functions as a suppressor via inhibition of ARA54-enhanced androgen receptor transactivation and prostate cancer cell growth

The androgen receptor (AR) requires coregulators for its optimal function. However, whether AR coregulators further need interacting protein(s) for their proper function remains unclear. Here we describe transgelin as the first ARA54-associated negative modulator for AR. Transgelin suppressed ARA54-enhanced AR function in ARA54-positive, but not in ARA54-negative, cells. Transgelin suppressed AR transactivation via interruption of ARA54 homodimerization and AR-ARA54 heterodimerization, resulting in the cytoplasmic retention of AR and ARA54. Stable transfection of transgelin in LNCaP cells suppressed AR-mediated cell growth and prostate-specific antigen expression, whereas this suppressive effect was abolished by the addition of ARA54-small interfering RNA. Results from tissue surveys showing decreased expression of transgelin in prostate cancer specimens further strengthened the suppressor role of transgelin. Our findings reveal the novel mechanisms of how transgelin functions as a suppressor to inhibit prostate cancer cell growth. They also demonstrate that AR coregulators, like ARA54, might have dual in vivo roles functioning as both a direct coactivator and as an indirect mediator in AR function. The finding that a protein can modulate AR function without direct interaction with AR might provide a new therapeutic approach, with fewer side effects, to battle prostate cancer by targeting AR indirectly.     

Loss of transgelin in breast and colon tumors and in RIE-1 cells by Ras deregulation of gene expression through Raf-independent pathways.

Activated Ras but not Raf can transform RIE-1 and other epithelial cells, indicating the critical importance of Raf-independent effector function in Ras transformation of epithelial cells. To elucidate the nature of these Raf-independent activities, we utilized representational difference analysis to identify genes aberrantly expressed by Ras through Raf-independent mechanisms in RIE-1 cells. We identified a total of 22 genes, both known and novel, whose expression was either activated or abolished by Ras but not Raf. The genes up-regulated encode proteins involved in protein or DNA synthesis, regulation of protease activity, or ligand binding, whereas those genes down-regulated encode actin cytoskeletal-, extracellular matrix-, and gap junction-associated proteins, and transmembrane receptor- or cytokine-like proteins. These results suggest that a key function of Raf-independent signaling involves deregulation of gene expression. We further characterized transgelin as a gene whose expression was abolished by Ras. Transgelin was identified previously as a protein whose expression was lost in virally transformed cell lines. We show that this loss is regulated at the level of gene expression and that both Raf-dependent and Raf-independent pathways are required to cause Ras down-regulation of transgelin in RIE-1 cells, whereas Raf alone is sufficient to cause its loss in NIH 3T3 fibroblasts. We also found that Ras-dependent and Ras-independent mechanisms can cause the down-regulation of transgelin in human breast and colon carcinoma cells lines and patient-derived tumor samples. We conclude that loss of transgelin gene expression may be an important early event in tumor progression and a diagnostic marker for breast and colon cancer development.     

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-α-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.     

Toll-like receptors 4 induces expression of matrix metalloproteinase-9 in human aortic smooth muscle cells

Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerotic lesions. It was confirmed that the presence of functional TLR4 promotes a proinflammatory phenotype and proliferation of vascular smooth muscle cells (VSMCs). Here we tested whether designed TLR4 small interfering RNAs (TLR4 siRNAs) is capable of inducing TLR4 deficient and simultaneously regulating the expression of matrix metalloproteinase-9 (MMP-9) in human aortic smooth muscle cells (HASMCs). Human aortic smooth muscle cells were obtained from Cascade Biologics (Portland, USA). The siRNAs used in this study were chemically synthesized by Ambion, diluted in RNase free water at concentration of 2 μg/ml. The TLR4 siRNAs were complexed with Lipofectamine^TM2000 in transfection buffer. After 30 min incubation at room temperature, the complexes were added to the cells. Subsequent to 5 h incubation, cells were treated with 10 ng/ml LPS for 24 h. RT–PCR analysis was used to detect mRNA expression of GAPDH, TLR4 and MMP-9; Western blot analysis was used to examine GAPDH, TLR4 and MMP-9 protein expression. It was shown that all three designed TLR4 siRNAs inhibited the expression of TLR4 in HASMCs as compared to nontargeting siRNA. Notably, TLR4 siRNA-1 exhibited the strongest inhibition effect. Transfection of HASMCs with TLR4 siRNA-1 resulted in down-regulation of LPS-induced expression of MMP-9. It was concluded that TLR4 siRNA-transfected HASMCs were capable for regulating the expression of MMP-9, providing support for the rational design of siRNAs as atherosclerotic therapy.     

Nicotine promotes atherosclerosis development in apolipoprotein E-deficient mice through α1-nAChR

α1 Nicotinic acetylcholine receptor (α1nAChR) is an important nicotine receptor that is widely distributed in vascular smooth muscle cells, macrophages, and endothelial cells. However, the role of α1nAChR in nicotine-mediated atherosclerosis remains unclear. The administration of nicotine for 12 weeks increased the area of the atherosclerotic lesion, the number of macrophages infiltrating the plaques, and the circulating levels of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α, in apolipoprotein E-deficient (ApoE^−/−) mice fed a high-fat diet. Nicotine also increased α1nAChR, calpain-1, matrix metalloproteinase-2 (MMP-2), and MMP-9 expression in the aortic tissue. Silencing of α1nAChR with an adenoassociated virus decreased the atherosclerotic size, lesion macrophage content, and circulating levels of inflammatory cytokines and suppressed α1nAChR, calpain-1, MMP-2, and MMP-9 expression in the nicotine group. In vitro, nicotine-induced α1nAChR, calpain-1, MMP-2, and MMP-9 expression in mouse vascular smooth muscle cells (MOVAS) and macrophages (RAW264.7), and enhanced the migration and proliferation of these cells. The silencing of α1nAChR inhibited these effects of nicotine MOVAS and RAW264.7 cells. Thus, we concluded that nicotine promoted the development of atherosclerosis partially by inducing the migration and proliferation of vascular smooth muscle cells and macrophages and inducing an inflammatory reaction. The effect of nicotine on atherogenesis may be mediated by α1nAChR-induced activation of the calpain-1/MMP-2/MMP-9 signaling pathway.     

Reduced peribronchial fibrosis in allergen-challenged MMP-9-deficient mice

Matrix metalloproteinases (MMPs) are a family of extracellular proteases that are responsible for the degradation of the extracellular matrix during tissue remodeling. We have used a mouse model of allergen-induced airway remodeling to determine whether MMP-9 plays a role in airway remodeling. MMP-9-deficient and wild-type (WT) mice were repetitively challenged intranasally with ovalbumin (OVA) antigen to develop features of airway remodeling including peribronchial fibrosis and increased thickness of the peribronchial smooth muscle layer. OVA-challenged MMP-9-deficient mice had less peribronchial fibrosis and total lung collagen compared with OVA-challenged WT mice. There was no reduction in mucus expression, smooth muscle thickness, or airway responsiveness in OVA-challenged MMP-9-deficient compared with OVA-challenged WT mice. OVA-challenged MMP-9-deficient mice had reduced levels of bronchoalveolar lavage (BAL) regulated on activation, normal T cell expressed, and secreted (RANTES), as well as reduced numbers of BAL and peribronchial eosinophils compared with OVA-challenged WT mice. There were no significant difference in levels of BAL eotaxin, thymus- and activation-regulated chemokine (TARC), or macrophage-derived chemokine (MDC) in OVA-challenged WT compared with MMP-9-deficient mice. Overall, this study demonstrates that MMP-9 may play a role in mediating selected aspects of allergen-induced airway remodeling (i.e., modest reduction in levels of peribronchial fibrosis) but does not play a significant role in mucus expression, smooth muscle thickness, or airway responsiveness.     

Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages

Matrix metalloproteinases (MMPs) are zinc endopeptidases that degrade extracellular matrix (ECM) components during normal and pathogenic tissue remodeling. Inappropriate expression of these enzymes contributes to the development of vascular pathology, including atherosclerosis. MMP-9 is expressed in its active form in atherosclerotic lesions and is believed to play an important role in vascular remodeling, smooth muscle cell migration, and plaque instability. We demonstrate here that the liver X receptors (LXRs) LXRalpha and LXRbeta inhibit basal and cytokine-inducible expression of MMP-9. Treatment of murine peritoneal macrophages with the synthetic LXR agonists GW3965 or T1317 reduces MMP-9 mRNA expression and blunts its induction by pro-inflammatory stimuli including lipopolysaccharide, interleukin-1beta, and tumor necrosis factor alpha. In contrast, macrophage expression of MMP-12 and MMP-13 is not altered by LXR ligands. We further show that the ability of LXR ligands to regulate MMP-9 expression is strictly receptor-dependent and is not observed in macrophages obtained from LXRalphabeta null mice. Analysis of the 5'-flanking region of the MMP-9 gene indicates that LXR/RXR heterodimers do not bind directly to the MMP-9 promoter. Rather, activation of LXRs represses MMP-9 expression, at least in part through antagonism of the NFkappaB signaling pathway. These observations identify the regulation of macrophage MMP-9 expression as a mechanism whereby activation of LXRs may impact macrophage inflammatory responses.     

ß-Catenin is markedly induced in a murine model of an arteriovenous fistula: the effect of metalloproteinase inhibition

Neointimal hyperplasia contributes to failure of hemodialysis arteriovenous fistulas (AVFs). Increased expression of matrix metalloproteinase (MMP)-9 occurs in AVFs, and MMP-9 is implicated in neointimal hyperplasia and vascular injury. Recent studies demonstrate that MMP-9, by degrading N-cadherin, leads to increased expression of β-catenin and β-catenin-dependent proliferation of smooth muscle cells. The present study examined this pathway in the venous limb of a murine AVF model. Western analyses demonstrate that, in this model, there is diminished expression of N-cadherin accompanied by increased expression of β-catenin, c-Myc, and proliferating cell nuclear antigen (PCNA). By immunohistochemistry, β-catenin and c-Myc localized to proliferating smooth muscle cells in the venous limb of the AVF. Increased expression of β-catenin was accompanied by augmented expression of phosphorylated (p)-glycogen synthase kinase (GSK)-3β, GSK-3β, and integrin-linked kinase. The administration of doxycycline suppressed MMP-9 expression but did not reduce venous histological injury in the AVF, or increase AVF patency assessed 6 wk after its creation. Doxycycline did not influence expression of β-catenin, c-Myc, GSK-3β, or integrin-linked kinase. Thus, in this vascular injury model, the upregulation of β-catenin cannot be readily attributed to MMP-9 upregulation; increased β-catenin expression may reflect either the upregulation of p-GSK-3β, GSK-3β, or integrin-linked kinase. This study provides the first exploration of β-catenin in an AVF, demonstrating substantial upregulation of this mitogenic signaling molecule and uncovering possible mechanisms that may account for such upregulation.     

Crystallization and preliminary crystallographic analysis of transgelin

Transgelin was solubly expressed in E. coli. Crystals of transgelin have been grown at 291K using sodium formate or PEG-4000 as precipitants. X-ray diffraction by the crystal extends to 2.3 A resolution. The crystal belongs to the space group P2(1), with the unit cell parameters a=39.3, b=61.9, c=56.0 A and beta=90.2 degrees .     

An elastinolytic enzyme detected in the culture medium of human arterial smooth muscle cells

The culture medium of human arterial smooth muscle cells exhibits an elastinolytic activity with 68 and 64 kDa on elastin substrate gels. The enzymatic activities are inhibited by ethylenediamine tetraacetic acid, a metalloproteinase inhibitor, but not by other inhibitors of serine, cysteine and aspartic proteinases. The proteinase in the culture medium is activatable by 4-aminophenylmercuric acetate and degrades insoluble elastin. Compared to other matrix metalloproteinases (MMP), the activity shows the similar elastinolytic pattern to that by MMP-2 purified from human rheumatoid synovium, while MMP-3 and MMP-9 have different lytic patterns and MMP-1 possesses no elastinolytic activity. An immunoblot analysis demonstrated that the 68-kDa enzyme is MMP-2. An immunofluorescence study illustrates that MMP-2 is localized within the cytoplasm of the smooth muscle cells. These findings suggest that the elastinolytic enzyme secreted by human arterial smooth muscle cells is MMP-2.