Involvement of Matricellular Proteins in Cellular Senescence: Potential Therapeutic Targets for Age-Related Diseases.

Senescence is a physiological and pathological cellular program triggered by various types of cellular stress. Senescent cells exhibit multiple characteristic changes. Among them, the characteristic flattened and enlarged morphology exhibited in senescent cells is observed regardless of the stimuli causing the senescence. Several studies have provided important insights into pro-adhesive properties of cellular senescence, suggesting that cell adhesion to the extracellular matrix (ECM), which is involved in characteristic morphological changes, may play pivotal roles in cellular senescence. Matricellular proteins, a group of structurally unrelated ECM molecules that are secreted into the extracellular environment, have the unique ability to control cell adhesion to the ECM by binding to cell adhesion receptors, including integrins. Recent reports have certified that matricellular proteins are closely involved in cellular senescence. Through this biological function, matricellular proteins are thought to play important roles in the pathogenesis of age-related diseases, including fibrosis, osteoarthritis, intervertebral disc degeneration, atherosclerosis, and cancer. This review outlines recent studies on the role of matricellular proteins in inducing cellular senescence. We highlight the role of integrin-mediated signaling in inducing cellular senescence and provide new therapeutic options for age-related diseases targeting matricellular proteins and integrins.

Bioactive TNIIIA2 Sequence in Tenascin-C Is Responsible for Macrophage Foam Cell Transformation; Potential of FNIII14 Peptide Derived from Fibronectin in Suppression of Atherosclerotic Plaque Formation.

One of the extracellular matrix proteins, tenascin-C (TN-C), is known to be upregulated in age-related inflammatory diseases such as cancer and cardiovascular diseases. Expression of this molecule is frequently detected, especially in the macrophage-rich areas of atherosclerotic lesions; however, the role of TN-C in mechanisms underlying the progression of atherosclerosis remains obscure. Previously, we found a hidden bioactive sequence termed TNIIIA2 in the TN-C molecule and reported that the exposure of this sequence would be carried out through limited digestion of TN-C by inflammatory proteases. Thus, we hypothesized that some pro-atherosclerotic phenotypes might be elicited from macrophages when they were stimulated by TNIIIA2. In this study, TNIIIA2 showed the ability to accelerate intracellular lipid accumulation in macrophages. In this experimental condition, an elevation of phagocytic activity was observed, accompanied by a decrease in the expression of transporters responsible for lipid efflux. All these observations were mediated through the induction of excessive ß1-integrin activation, which is a characteristic property of the TNIIIA2 sequence. Finally, we demonstrated that the injection of a drug that targets TNIIIA2's bioactivity could rescue mice from atherosclerotic plaque expansion. From these observations, it was shown that TN-C works as a pro-atherosclerotic molecule through an internal TNIIIA2 sequence. The possible advantages of clinical strategies targeting TNIIIA2 are also indicated.

Heat shock-induced heme oxygenase-1 expression in a mouse hepatoma cell line is dependent on HSF1 and modified by NRF2 and BACH1.

The induction mechanism of heme oxygenase-1 (HO-1) by heat shock (HS) is still unknown. Here, we discovered that HS activates the HO-1 expression in a mouse hepatoma cell line (Hepa 1-6). Knockdown experiments showed that the HS-induced HO-1 expression was dependent on HS factor 1 (HSF1). A chromatin immunoprecipitation (ChIP) assay demonstrated that the HS-activated HSF1 bound to the HS elements (HSEs) in the upstream enhancer 1 region (E1). Unexpectedly, HS also facilitates the BTB and CNC homology 1 (BACH1) binding to the Maf recognition elements (MAREs) in E1. We examined the effects of a catalytically inactive CRISPR-associated 9 nucleases (dCas9) with short guide RNAs (sgRNAs), and demonstrated that the HSF1 binding to HSEs in E1 was indispensable for the HS-induced HO-1 expression. Heme treatment (HA) dissociates BACH1 from MAREs and facilitated the binding of nuclear factor-erythroid-2-related factor 2 (NRF2) to MAREs. Following treatment with both HS and HA, the HO-1 induction and the HSF1 binding to HSEs in E1 were most notably observed. These results indicate that the HS-induced HO-1 expression is dependent on the HSF1 binding to HSEs in E1, although modulated by the BACH1 and NRF2 binding to MAREs within the same E1.

Anoikis resistance conferred by tenascin-C-derived peptide TNIIIA2 and its disruption by integrin inactivation.

Glioblastoma multiforme (GBM), the most common brain tumor in adults, has an extremely poor prognosis, which is attributed to the aggressive properties of GBM cells, such as dysregulated proliferation and disseminative migration. We recently found that peptide TNIIIA2, derived from tenascin-C (TNC), which is highly expressed in GBM, contributes to the acquisition of these aggressive properties through ß1-integrin activation. In general, cancer cells often acquire an additional malignant property that confers resistance to apoptosis due to loss of adhesion to the extracellular matrix, termed anoikis resistance. Our present results show that regulation of ß1-integrin activation also plays a key role in both the development and loss of anoikis resistance in GBM cells. Despite being derived from a GBM with an extremely poor prognosis, the human GBM cell line T98G was susceptible to anoikis but became anoikis resistant via treatment with peptide TNIIIA2, which is able to activate ß1-integrin. The TNIIIA2-conferred anoikis resistance of T98G cells was disrupted by further addition of peptide FNIII14, which has the ability to inactivate ß1-integrin. Moreover, anchorage-independent survival of GBM cells in suspension culture was abrogated by peptide FNIII14, but not by RGD and CS-1 peptides, which are antagonistic for integrins α5ß1, αvß3, and α4ß1. These results suggest that GBM cells develop anoikis resistance through activation of ß1-integrin by TNC-derived peptide TNIIIA2, which is abundantly released into the tumor microenvironment of GBM. Inactivation of ß1-integrin may provide a promising strategy to overcome the apoptosis resistance of cancer cells, including GBM.

Involvement of Integrin-Activating Peptides Derived from Tenascin-C in Cancer Aggression and New Anticancer Strategy Using the Fibronectin-Derived Integrin-Inactivating Peptide.

Matricellular proteins, which exist in association with the extracellular matrix (ECM) and ECM protein molecules, harbor functional sites within their molecular structures. These functional sites are released through proteolytic cleavage by inflammatory proteinases, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and the peptides containing these functional sites have unique biological activities that are often not detected in the parent molecules. We previously showed that tenascin-C (TNC) and plasma fibronectin (pFN), examples of matricellular proteins, have cryptic bioactive sites that have opposite effects on cell adhesion to the ECM. A peptide containing the bioactive site of TNC, termed TNIIIA2, which is highly released at sites of inflammation and in the tumor microenvironment (TME), has the ability to potently and persistently activate ß1-integrins. In the opposite manner, the peptide FNIII14 containing the bioactive site of pFN has the ability to inactivate ß1-integrins. This review highlights that peptide TNIIIA2 can act as a procancer factor and peptide FNIII14 can act as an anticancer agent, based on the regulation on ß1-integrin activation. Notably, the detrimental effects of TNIIIA2 can be inhibited by FNIII14. These findings open the possibility for new therapeutic strategies based on the inactivation of ß1-integrin by FNIII14.

Peptide TNIIIA2 Derived from Tenascin-C Contributes to Malignant Progression in Colitis-Associated Colorectal Cancer via ß1-Integrin Activation in Fibroblasts.

Inflammatory bowel diseases increase the risk of colorectal cancer and colitis-associated colorectal cancer (CAC). Tenascin-C, a matricellular protein, is highly expressed in inflammatory bowel diseases, especially colorectal cancer. However, the role of tenascin-C in the development of CAC is not yet fully understood. We previously showed that a peptide derived from tenascin-C, peptide TNIIIA2, induces potent and sustained activation of ß1-integrin. Moreover, we recently reported that peptide TNIIIA2 promotes invasion and metastasis in colon cancer cells. Here, we show the pathological relevance of TNIIIA2-related functional site for the development of CAC. First, expression of the TNIIIA2-containing TNC peptides/fragments was detected in dysplastic lesions of an azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model. In vitro experiments demonstrated that conditioned medium from peptide TNIIIA2-stimulated human WI-38 fibroblasts induced malignant transformation in preneoplastic epithelial HaCaT cells. Indeed, these pro-proliferative effects stimulated by peptide TNIIIA2 were abrogated by peptide FNIII14, which has the ability to inactivate ß1-integrin. Importantly, peptide FNIII14 was capable of suppressing polyp formation in the AOM/DSS model. Therefore, tenascin-C-derived peptide TNIIIA2 may contribute to the formation of CAC via activation of stromal fibroblasts based on ß1-integrin activation. Peptide FNIII14 could represent a potential prophylactic treatment for CAC.

Autocrine Production of PDGF Stimulated by the Tenascin-C-Derived Peptide TNIIIA2 Induces Hyper-Proliferation in Glioblastoma Cells.

Expression level of tenascin-C is closely correlated to poor prognosis in glioblastoma patients, while the substantial role of tenascin-C responsible for aggressive progression in glioblastoma cells has not been clarified. We previously found that peptide TNIIIA2, which is derived from the tumor-associated tenascin-C variants, has the ability to promote cell adhesion by activating ß1-integrins. Our recent study demonstrated that potentiated activation of integrin α5ß1 by TNIIIA2 causes not only a dysregulated proliferation in a platelet-derived growth factor (PDGF)-dependent manner, but also disseminative migration in glioblastoma cells. Here, we show that TNIIIA2 enhances the proliferation in glioblastoma cells expressing PDGF-receptorß, even without exogenous PDGF. Mechanistically, TNIIIA2 induced upregulated expression of PDGF, which in turn stimulated the expression of tenascin-C, a parental molecule of TNIIIA2. Moreover, in glioblastoma cells and rat brain-derived fibroblasts, tenascin-C upregulated matrix metalloproteinase-2, which has the potential to release TNIIIA2 from tenascin-C. Thus, it was shown that autocrine production of PDGF triggered by TNIIIA2 functions to continuously generate a functional amount of PDGF through a positive spiral loop, which might contribute to hyper-proliferation in glioblastoma cells. TNIIIA2 also enhanced in vitro disseminative migration of glioblastoma cells via the PKCα signaling. Collectively, the tenascin-C/TNIIIA2 could be a potential therapeutic target for glioblastoma.

The Promoting Effect of the Extracellular Matrix Peptide TNIIIA2 Derived from Tenascin-C in Colon Cancer Cell Infiltration.

The extracellular matrix (ECM) molecule tenascin C (TNC) is known to be highly expressed under various pathological conditions such as inflammation and cancer. It has been reported that the expression of TNC is correlated with the malignant potential of cancer. In our laboratory, it was found that the peptide derived from the alternative splicing domain A2 in TNC, termed TNIIIA2, has been shown to influence a variety of cellular processes, such as survival, proliferation, migration, and differentiation. In this study, we investigated the effect of TNC/TNIIIA2 on the invasion and metastasis of colon cancer cells, Colon26-M3.1, or PMF-Ko14, using an in vitro and in vivo experimental system. The degree of cell invasion was increased by the addition of TNC and TNIIIA2 in a dose-dependent manner. The invasion by TNC and TNIIIA2 were suppressed by an MMP inhibitor or TNIIIA2-blocking antibody. In an in vivo experiment, pulmonary metastasis was promoted conspicuously by the addition of TNIIIA2. In this study, we found that colon cancer cell invasion and metastasis was accelerated by TNC/TNIIIA2 via MMP induction. This result suggests the possibility of a new strategy targeting TNC/TNIIIA2 for colon cancer.

A novel mechanism underlying the basic defensive response of macrophages against Mycobacterium infection.

Following inhalation of Mycobacterium tuberculosis, including bacillus Calmette-Guérin (BCG), pathogens enter and grow inside macrophages by taking advantage of their phagocytic mechanisms. Macrophages often fail to eliminate intracellular M. tuberculosis, leading to the induction of host macrophage death. Despite accumulating evidence, the molecular mechanisms underlying M. tuberculosis infection-induced cell death remain controversial. In this study, we show the involvement of two distinct pathways triggered by TLR2 and ß2 integrin in BCG infection-induced macrophage apoptosis. First, BCG infection induced activation of ERK1/2, which in turn caused phosphorylation/activation of the proapoptotic protein Bim in mouse macrophage-like Raw 264.7 cells. BCG-infected Raw cells treated with U0126, an MEK/ERK inhibitor, led to the suppression of Bim phosphorylation alongside a remarkable increase in the number of viable macrophages. Small interfering RNA-mediated knockdown of Bim rescued the macrophages from the apoptotic cell death induced by BCG infection. Stimulation with Pam3CSK, a TLR2 agonist, induced macrophage apoptosis with a concomitant increase in the phosphorylation/activation of MEK/ERK and Bim. These observations indicate the important role of the TLR2/MEK/ERK/Bim pathway in BCG infection-induced macrophage apoptosis. Second, we used the ß2 integrin agonists C3bi and fibronectin to show that the ß2 integrin-derived signal was involved in BCG infection-induced apoptosis, independent of MEK/ERK activation. Interestingly, latex beads coated with Pam3CSK and C3bi were able to induce apoptosis in macrophages to the same extent and specificity as that induced by BCG. Taken together, two distinct pattern-recognition membrane receptors, TLR2 and ß2 integrin, acted as triggers in BCG infection-induced macrophage apoptosis, in which MEK/ERK activation played a crucial role following the engagement of TLR2.

Tenascin-C-derived peptide TNIIIA2 highly enhances cell survival and platelet-derived growth factor (PDGF)-dependent cell proliferation through potentiated and sustained activation of integrin α5ß1.

Tenascin-C is an adhesion modulatory matrix protein that is highly expressed in tumors; however, its biochemical activity involved in tumorigenesis is not fully understood. On the other hand, increasing evidence indicates the importance of integrin α5ß1 in cancer development. We previously demonstrated that tenascin-C harbors a functional site that can be released as a proadhesive peptide such as TNIIIA2. Peptide TNIIIA2 is capable of inducing activation of ß1-integrins including α5ß1 via syndecan-4. In this study the proadhesive effect of TNIIIA2 was characterized by potentiated and sustained activation of integrin α5ß1. Based on this effect, TNIIIA2 rendered nontransformed fibroblasts (NIH3T3) resistant to serum deprivation-elicited anoikis through activation of the Akt/Bcl-2 pathway. Moreover, TNIIIA2 hyperstimulated PDGF-dependent proliferation of NIH3T3 by activating integrin α5ß1. Tenascin-C, a parental protein of TNIIIA2, also stimulated PDGF-dependent proliferation, which was blocked by a matrix metalloproteinase-2/9 inhibitor and an anti-TNIIIA2 function-blocking antibody, suggesting proteolytic exposure of the proadhesive effect of TNIIIA2. Mechanistic analyses revealed that TNIIIA2 induced a lateral association of PDGF receptor ß with the molecular complex of activated integrin α5ß1 and syndecan-4 in the membrane microdomains enriched with cholesterol/caveolin-1, resulting in prolonged activation of PDGF receptor ß and the subsequent Ras/mitogen-activated protein kinase pathway in a PDGF-dependent manner. Of note, TNIIIA2 induced continuous proliferation in NIH3T3 in an integrin α5ß1-dependent manner even after they formed a confluent monolayer. Thus, it was proposed that tenascin-C might be involved in deregulated cell growth through potentiated and sustained activation of integrin α5ß1 after exposure of the proadhesive effect of TNIIIA2.

Increased SPARC in brain microvessels of ob/ob mice accelerates molecular transport into the brain accompany with albumin.

Cytotoxic metabolites originating from the peripheral circulation can induce central nervous system complications associated with diabetes. Since a large proportion of these metabolites bind to plasma albumin, mechanisms for transporting albumin-metabolite complexes into the brain exist under diabetic conditions. Secreted protein acidic and rich in cysteine (SPARC) is one of the vesicular transport receptors responsible for albumin transport. This study aimed to investigate the changes in SPARC expression and cellular albumin transfer under high-glucose conditions and evaluate the permeability of molecules with high protein-bound properties to the brain tissue. Glucose (30 mM) increased SPARC expression, and intracellular albumin accumulation in NIH3T3 cells. In addition, these changes were observed in the brain of ob/ob mice. Brain microvessels function as a physiological barrier to limit the penetration of molecules from the peripheral blood circulation into the brain by forming tight junctions. Although protein expression of molecules involved in tight junction formation and cell adhesion was increased in the brain microvessels of ob/ob mice, molecular transfer into the brain through cellular junctions was not enhanced. However, Evans blue dye injected into the peripheral vein and endogenous advanced glycation end-products, exerted a high protein-binding property and accumulated in their brains. These observations indicate that peripheral molecules with high protein-binding properties invade the brain tissue and bind to albumin through transcytosis mediated by SPARC.

Lysophosphatidic acid induces early growth response-1 (Egr-1) protein expression via protein kinase Cδ-regulated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activation in vascular smooth muscle cells.

Lysophosphatidic acid (LPA) modulates vascular cell function in vitro and in vivo via regulating the expression of specific genes. Previously, we reported that a transcriptional mechanism controls LPA-induced expression of Egr-1 in vascular smooth muscle cells. Egr-1 is a master transcription factor mediating the expression of various genes that have been implied to modulate a broad spectrum of vascular pathologies. In this study, we determined the essential intracellular signaling pathway leading to LPA-induced Egr-1 expression. Our data demonstrate that activation of ERK1/2 and JNK, but not p38 MAPK, is required for LPA-induced Egr-1 expression in smooth muscle cells. We provide the first evidence that MEK-mediated JNK activation leads to LPA-induced gene expression. JNK2 is required for Egr-1 induction. Examining the upstream kinases that mediate ERK and JNK activation, leading to Egr-1 expression, we found that LPA-induced activation of MAPKs and expression of Egr-1 are dependent on PKC activation. We observed that LPA rapidly activates PKCδ and PKCθ. Overexpression of dominant-negative PKCδ, but not dominant-negative PKCθ, diminished activation of ERK and JNK and blocked LPA-induced expression of Egr-1 mRNA and protein. We also evaluated LPA receptor involvement. Our data reveal an intracellular regulatory mechanism: LPA induction of Egr-1 expression is via LPA cognate receptor (LPA receptor 1)-dependent and PKCδ-mediated ERK and JNK activation. This study provides the first evidence that PKCδ mediates ERK and JNK activation in the LPA signaling pathway and that this pathway is required for LPA-induced gene regulation as evidenced by Egr-1 expression.

Eukaryotic translation elongation factor 1A induces anoikis by triggering cell detachment.

Anoikis, apoptosis because of loss of cell anchorage, is crucial for tissue homeostasis. Fibronectin not only provides a scaffold for cell anchorage but also harbors a cryptic antiadhesive site capable of inducing ß1-integrin inactivation. In this study, this cryptic antiadhesive site is implicated in spontaneous induction of anoikis. Nontransformed fibroblasts (NIH3T3) adhering to a fibronectin substratum underwent anoikis during serum starvation culture. This anoikis was caused by proteolytic exposure of the cryptic antiadhesive site in fibronectin by matrix metalloproteinase. Eukaryotic elongation factor 1A (eEF1A) was identified as a membrane receptor for the exposed antiadhesive site. Serum starvation raised the membrane residence of eEF1A, and siRNA-based disruption of this increase rendered cells anoikis-resistant. By contrast, cells became more susceptible to anoikis in parallel with increased membrane residence of eEF1A by enforced expression. These results demonstrate that eEF1A acts as a membrane receptor for the cryptic antiadhesive site of fibronectin, which contributes to cell regulation, including anoikis, through negative regulation of cell anchorage.

Preferential involvement of Na⁺/Ca²âº exchanger type-1 in the brain damage caused by transient focal cerebral ischemia in mice.

The Na(+)/Ca(2+) exchanger (NCX), an ion-transporter located in the plasma membrane of neuronal cells, contributes to intracellular Ca(2+) homeostasis. Within the brain, three isoforms (NCX1, NCX2, and NCX3) are widely distributed. However, it is not clear to what extent these isoforms are involved in ischemic brain damage in mammals. We therefore used genetically altered mice and isoform-selective NCX inhibitors in a model of transient focal ischemia to investigate the role of each NCX isoform in ischemic brain damage. NCX isoform-mutant mice (NCX1(+/-), NCX2(+/-), and NCX3(+/-)) and wild-type mice were subjected to 90min of middle cerebral artery occlusion (MCAO) followed by 24h of reperfusion. One of three NCX inhibitors [SN-6, KB-R7943, or SEA0400 (3 or 10mgkg(-1), i.p.)] was administered to ddY mice at 30min before more prolonged (4-h) MCAO followed by 24h of reperfusion. After transient MCAO reperfusion, the cerebral infarcts in NCX1(+/-) mice, but not those in NCX2(+/-) or NCX3(+/-) mice, were significantly smaller than those in wild-type mice. SN-6 and SEA0400, which are more selective for the NCX1 isoform, significantly reduced the infarct volume at 10mg/kg. In contrast, KB-R7943, which is more selective for NCX3, did not. These results suggest that the NCX1 isoform may act preferentially (vs. the NCX2 and NCX3 isoforms) to exacerbate the cerebral damage caused by ischemic insult in mice, and that NCX1-selective inhibitors warrant investigation as a potential therapeutic agents for stroke.

The cell adhesion and proliferation activities of a peptide derived from human tenascin-C are dependent on two Ile residues.

A tenascin-C derived peptide (TNIIIA2 peptide, 1) stimulated ß1 integrin-mediated cell adhesion via binding to syndecan-4. Ala-substituted peptides were synthesized to understand the structure-activity relationship. Peptides in which basic amino acids were substituted showed reduced cell adhesion activity, but their proliferation activities were similar to or higher than those mediated by peptide 1. In contrast, peptides in which the Ile residues of peptide 1 were replaced were inactive, indicating that the Ile residues are critical for the peptide's activity. CD analysis suggested that the Ile residues are necessary for the formation of a specific conformation required for binding to syndecan-4.

New molecular mechanisms for cardiovascular disease: cardiac hypertrophy and cell-volume regulation.

Cardiac hypertrophy is an increase in the muscle volume of the ventricle due to the enlargement of cardiac cells. Physiological cardiac hypertrophy is the normal response to healthy exercise, and pathological hypertrophy is the response to increased stress such as hypertension. Intracellular and extracellular aniosmotic conditions also change cell volume. Since persistent cell swelling or cell shrinkage during aniosmotic conditions results in cell death, the ability to regulate cell volume is important for the maintenance of cellular homeostasis. Cell swelling activates a regulatory volume decrease (RVD) response in which solute leakage pathways are stimulated and solute with water exits cells, reducing the cell volume towards the original value. In cardiac cells, one of the essential factors for cell-volume regulation is the volume-regulated anion channel (VRAC). However, the relationship between cardiac hypertrophy and cell-volume regulation is not clear. In this review, we introduce our recent findings showing that the impairment of VRAC current is exhibited in ventricular cells from mice with cardiac hypertrophy induced by transverse aortic constriction. Similar results were shown in caveolin-3-deficient mice, which develop cardiac hypertrophy without pressure overload. These results suggest that VRAC will be a new target for protection from the development of cardiac hypertrophy.

Wound Healing Promotion by Hyaluronic Acid: Effect of Molecular Weight on Gene Expression and In Vivo Wound Closure.

Hyaluronic acid (HA) has been known to play an important role in wound healing process. However, the effect of molecular weight (MW) of exogenously administered HA on the wound healing process has not been fully understood. In this study, we investigated HA with different MWs on wound healing process using human epidermal keratinocytes and dermal fibroblasts. Cell proliferation and migration ability were assessed by water soluble tetrazolium (WST) assay and wound scratch assay. We examined the effect of HA addition in a full-thickness wound model in mice and the gene expression related to wound healing. Proliferation and migration of HaCaT cells increased with the increase of MW and concentration of HA. Interleukin (IL-1ß), IL-8 and vascular endothelial growth factor (VEGF) as well as matrix metalloproteinase (MMP)-9 and MMP-13 were significantly upregulated by high molecular weight (HMW) HA in keratinocytes. Together with VEGF upregulation and the observed promotion of HaCaT migration, HA with the MW of 2290 kDa may hold potential to improve re-epithelialization, a critical obstacle to heal chronic wounds.

Induction of cellular senescence in fibroblasts through ß1-integrin activation by tenascin-C-derived peptide and its protumor effect.

Tenascin-C is upregulated during inflammation and tumorigenesis, and its expression level is correlated with a poor prognosis in several malignancies. Nevertheless, the substantial role of tenascin-C in cancer progression is poorly understood. Previously, we found that a peptide derived from tenascin-C, termed TNIIIA2, acts directly on tumor cells to activate ß1-integrin and induce malignant progression. Here, we show that ß1-integrin activation by TNIIIA2 in human fibroblasts indirectly contributes to cancer progression through the induction of cellular senescence. Prolonged treatment of fibroblasts with TNIIIA2 induced cellular senescence, as characterized by the suppression of cell growth and the induction of senescence-associated-ß-galactosidase and p16INK4a expression. The production of reactive oxygen species and subsequent DNA damage were responsible for the TNIIIA2-induced senescence of fibroblasts. Interestingly, peptide FNIII14, which inactivates ß1-integrin, inhibited fibroblast senescence induced not only by TNIIIA2 but also by H2O2, suggesting that ß1-integrin activation plays a critical role in the induction of senescence in fibroblasts. Moreover, TNIIIA2-induced senescent fibroblasts secreted heparin-binding epidermal growth factor-like growth factor (HB-EGF), which caused preneoplastic epithelial HaCaT cells to acquire malignant properties, including colony-forming and focus-forming abilities. Thus, our study demonstrates that tenascin-C-derived peptide TNIIIA2 induces cellular senescence in fibroblasts through ß1-integrin activation, causing cancer progression via the secretion of humoral factors such as HB-EGF.

Biologically Active TNIIIA2 Region in Tenascin-C Molecule: A Major Contributor to Elicit Aggressive Malignant Phenotypes From Tumors/Tumor Stroma.

Tenascin (TN)-C is highly expressed specifically in the lesions of inflammation-related diseases, including tumors. The expression level of TN-C in tumors and the tumor stroma is positively correlated with poor prognosis. However, no drugs targeting TN-C are currently clinically available, partly because the role of TN-C in tumor progression remains controversial. TN-C harbors an alternative splicing site in its fibronectin type III repeat domain, and its splicing variants including the type III-A2 domain are frequently detected in malignant tumors. We previously identified a biologically active region termed TNIIIA2 in the fibronectin type III-A2 domain of TN-C molecule and showed that this region is involved in promoting firm and persistent cell adhesion to fibronectin. In the past decade, through the exposure of various cell lines to peptides containing the TNIIIA2 region, we have published reports demonstrating the ability of the TNIIIA2 region to modulate distinct cellular activities, including survival/growth, migration, and invasion. Recently, we reported that the signals derived from TNIIIA2-mediated ß1 integrin activation might play a crucial role for inducing malignant behavior of glioblastoma (GBM). GBM cells exposed to the TNIIIA2 region showed not only exacerbation of PDGF-dependent proliferation, but also acceleration of disseminative migration. On the other hand, we also found that the pro-inflammatory phenotypic changes were promoted when macrophages are stimulated with TNIIIA2 region in relatively low concentration and resulting MMP-9 upregulation is needed to release of the TNIIIA2 region from TN-C molecule. With the contribution of TNIIIA2-stimulated macrophages, the positive feedback spiral loop, which consists of the expression of TN-C, PDGF, and ß1 integrin, and TNIIIA2 release, seemed to be activated in GBM with aggressive malignancy. Actually, the growth of transplanted GBM grafts in mice was significantly suppressed via the attenuation of ß1 integrin activation. In this review, we thus introduce that the TNIIIA2 region has a significant impact on malignant progression of tumors by regulating cell adhesion. Importantly, it has been demonstrated that the TNIIIA2 region exerts unique biological functions through the extremely strong activation of ß1-integrins and their long-lasting duration. These findings prompt us to develop new therapeutic agents targeting the TNIIIA2 region.

Exposure of the cryptic de-adhesive site FNIII14 in fibronectin molecule and its binding to membrane-type eEF1A induce migration and invasion of cancer cells via ß1-integrin inactivation.

Cell migration is a highly coordinated process that involves not only integrin-mediated adhesion but also de-adhesion. We previously found that a cryptic de-adhesive site within fibronectin molecule, termed FNIII14, weakens cell adhesion to the extracellular matrix by inactivating ß1-integrins. Surprisingly, eukaryotic translation elongation factor-1A (eEF1A), an essential factor during protein biosynthesis, was identified as a membrane receptor that mediates the de-adhesive effect of FNIII14. Here, we demonstrate that FNIII14-mediated de-adhesion causes enhanced migration and invasion in two types of highly invasive/metastatic cancer cells, resulting in the initiation of metastasis. Both in vitro migration and invasion of highly invasive human melanoma cell line, Mum2B, were inhibited by a matrix metalloproteinase (MMP)-2/9 inhibitor or a function-blocking antibody against FNIII14 (anti-FNIII14 Ab), suggesting that MMP-mediated exposure of the cryptic de-adhesive site FNIII14 was responsible for Mum2B cell migration and invasion. The MMP-induced FNIII14 exposure was also shown to be functional in the migration and invasion of highly metastatic mouse breast cancer cell line 4T1. Overexpression and knockdown experiments of eEF1A in Mum2B cells revealed that the migration and invasion were dependent on the membrane levels of eEF1A. In vivo experiments using tumor xenograft mouse models derived from Mum2B and 4T1 cell lines showed that the anti-FNIII14 Ab has a significant anti-metastatic effect. Thus, these results provide novel insights into the regulation of cancer cell migration and invasion and suggest promising targets for anti-metastasis strategies.