Expression of Integrin α6β1 Enhances Tumorigenesis in Glioma Cells

The integrin α6β1 and its main ligand laminin-111 are overexpressed in glioblastoma, as compared with normal brain tissue, suggesting they may be involved in glioblastoma malignancy. To address this question, we stably expressed the α6 integrin subunit in the U87 cell line via retroviral-mediated gene transfer. We show that cell surface expression of the α6β1 integrin led to dramatic changes in tumor U87 cell behavior, both in vitro and in vivo. Nude mice receiving either subcutaneous or intracerebral inoculation of α6β1-expressing cells developed substantially more voluminous tumors than mice injected with control cells. The difference in tumor growth was associated with a marked increase in vascularization in response to α6β1 integrin expression and may also be related to changes in the balance between cell proliferation and survival. Indeed, expression of α6β1 enhanced proliferation and decreased apoptosis of U87 cells both in the tumor and in vitro. Additionally, we demonstrate that α6β1 is implicated in glioblastoma cell migration and invasion and that laminin-111 might mediate dissemination of α6β1-positive cells in vivo. Our results highlight for the first time the considerable role of the integrin α6β1 in glioma progression.Malignant brain tumors have an increasing incidence in both children and adults. In adults, the most common type of primary brain tumor, malignant glioma, is considered as one of the deadliest of human cancers. Despite recent advances in both diagnostic modalities and therapeutic strategies, the 5-year survival rate of less than 3% in patients with glioblastoma is among the lowest for all cancers.¹ Patients with the most malignant histopathological subtype, glioblastoma, carry the worst prognosis, with median survival rate of less than 1 year, despite aggressive surgery associated with adjuvant radiotherapy and chemotherapy.¹ Glioblastoma are characterized by rapidly dividing cells, high degree of vascularity, invasion into normal brain tissue, and an intense resistance to death-inducing stimuli.^2,3 Since integrins, the major family of extracellular matrix (ECM) receptors, are involved in these events, they are one of the most promising molecules to consider for a targeted therapy.Integrins are cell surface transmembrane αβ heterodimers that recognize specific ECM ligands. The combination of α and β subunits, leading to the formation of at least 24 receptors, determines the ligand specificity.⁴ Glioblastoma commonly displays enhanced expression of several integrins along with their ECM ligands: αvβ3 and αvβ5 (tenascin and vitronectin receptors), α5β1 (fibronectin receptor), α2β1 (collagens receptor), and α3β1, α6β4, and α6β1 (laminins receptors).⁵ Numerous studies have focused on the αv integrin family. The integrins αvβ3 and αvβ5 are markers of glioblastoma malignancy⁶ and influence a variety of processes in glioblastoma progression in vivo, including proliferation, apoptosis, and angiogenesis.⁷ Furthermore, cilengitide, an αvβ3 and αvβ5 integrins antagonist, extends mouse survival by delaying the tumor growth^8,9 and is nowadays in clinical trial for recurrent malignant glioma. Two other integrins, α5β1 and α3β1, have been shown to be implicated in glioma cell adhesion and migration in vitro.^10,11 In addition, the use of α5β1 antagonists reduces glioma cell proliferation in vitro,¹⁰ while α3β1 antagonists inhibited glioma invasion in vivo.¹¹The α6 integrin subunit associates with β1 or β4 subunits to form functional heterodimers that selectively bind laminins. The α6β4 integrin is essential for the organization and maintenance of epithelial hemidesmosomes that link the intermediate filaments with the extracellular matrix.¹² The major ligand of α6β4 is the laminin-332, while α6β1 is a well-characterized laminin-111 receptor. Overexpression of α6β1 integrin has been associated with the progression of many epithelial tumors. In particular, induction of α6β1 expression is an early event in hepatocellular carcinogenesis.^13,14 In the same way, during prostate cancer progression α6β1 is continually expressed and found in micrometastases.¹⁵ Expression of α6β1 integrin has also been linked to metastatic potential of melanoma cells,¹⁶ and has been involved in the survival and metastatic potential of human breast carcinoma cells.^17,18 Moreover, in a recent study using the α6-blocking antibody GoH3, Lee et al¹⁹ inhibited angiogenesis and breast carcinoma growth in vivo.Several studies concerning gliomas and the α6β1 ligand laminin-111 have been reported in the literature. Using immunohistochemistry studies, Gingras et al²⁰ showed that α6 integrin was strongly expressed in glioblastoma tissue, whereas it was weakly expressed in normal brain. Previtali et al²¹ confirmed that the expression of α6 was increased in glioblastoma and in other central nervous system tumors, such as meningioma, astrocytoma, and neuroblastoma, when compared with the autologous normal tissue counterpart. In glioblastoma biopsies, laminin-111 is highly expressed on tumor blood vessels, but also within the brain tumor as punctuate deposits and at the tumor invasion front.²² In vitro, glioma cells can both secrete laminin-111 and induce its expression in normal brain tissue.^22,23,24 Moreover, laminin-111 is one of the most permissive substrates for adhesion and migration of glioma cells in vitro.^25,26,27 Additionally, over laminin-111, migrating glioma cells are protected from apoptosis.²⁸ For all these reasons, we hypothesized that laminin-111 and its main receptor α6β1 may contribute to glioblastoma progression.In the present study we investigated the role of integrin α6β1 in glioblastoma malignancy by using U87, a well-characterized glioblastoma cell line. We report that stable expression of α6β1 in this α6-negative cell line leads to enhanced tumor progression and tumor growth in vivo. We demonstrate that α6β1 is pro-angiogenic and acts on the balance between proliferation and apoptosis. Additionally, we show that α6β1 is involved in glioblastoma cell migration and invasion. Our results highlight for the first time the considerable role of integrin α6β1 in the malignant phenotype of glioblastoma cells and demonstrate that the α6β1-expressing cell is an appropriate model for the study of glioblastoma progression.     

Transgenic expression of Laminin α1 chain does not prevent muscle disease in the mdx mouse model for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder, and one of the most frequently encountered, but one for which there is as yet no treatment. Laminin-111 protein therapy was recently shown to be a promising approach to prevent muscle disease in the mdx mouse model of DMD. The present study demonstrated that transgenic expression of laminin α1 chain in mdx animals, resulting in laminin-111 heterotrimer formation in mdx muscle, does not improve the dystrophic phenotype. The mdx mice overexpressing laminin-111 (mdxLMα1) display features of mdx littermates: dystrophic pattern of muscle biopsy, elevated creatine kinase levels, reduced muscle strength, and decreased sarcolemmal integrity. Increased expression of integrin α7 is not beneficial for mdxLMα1 muscle, and components of the dystrophin-glycoprotein complex are not restored at the sarcolemma on laminin-111 overexpression. In summary, further studies are needed to verify the functionality of laminin-111 protein therapy in DMD and to describe the molecular events resulting from this approach.     

Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack α7 Integrin and Utrophin

The α7β1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of α7β1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and α7 integrin (mdx/α7^−/−), or dystrophin and utrophin (mdx/utr^−/−), exhibit severe muscle pathology and die prematurely. To explore the contribution of the α7β1 integrin and utrophin to muscle integrity and function, we generated mice lacking both α7 integrin and utrophin. Surprisingly, mice that lack both α7 integrin and utrophin (α7/utr^−/−) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to α7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in α7/utr^−/− skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The α7/utr^−/− mouse, therefore, demonstrates the critical roles of α7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the α7β1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease that affects 1 in every 3500 live male births. Patients with DMD have impaired mobility, are restricted to a wheelchair by their teens, and die from cardiopulmonary failure in their early twenties.^1,2 Currently, there is no cure or effective treatment for this devastating disease. Mutations in the dystrophin gene resulting in loss of the dystrophin protein are the cause of disease in DMD patients and the mdx mouse model.^3,4,5,6,7The dystrophin glycoprotein complex links laminin in the extracellular matrix to the actin cytoskeleton. The N-terminal region of dystrophin interacts with cytoskeletal F-actin⁸ and the C-terminal region associates with the dystrophin-associated protein complex, which include α- and β-dystroglycan, α- and β-syntrophin, the sarcoglycans, and sarcospan.⁹ In DMD, the absence of dystrophin leads to disruption of the dystrophin glycoprotein complex, resulting in increased muscle fragility and altered cell signaling.⁹ Loss of this critical transmembrane linkage complex in DMD patients and mdx mice results in progressive muscle damage and weakness, inflammation, necrosis, and fibrosis. Lack of dystrophin also leads to abnormalities at myotendinous and neuromuscular junctions (MTJ and NMJ), which further contribute to skeletal muscle damage.^{10,11,12,13,14,15,16,17} In addition, defective muscle repair in DMD patients eventually results in muscle degeneration exceeding the rate of regeneration.¹⁸ Overall, dystrophin is critical for muscle function, structure, and stability, and its absence results in progressive muscle wasting and severe muscular dystrophy. In the absence of dystrophin two additional laminin-binding receptors, the α7β1 integrin and utrophin, are up-regulated in the skeletal muscle of DMD patients and mdx mice, which may compensate for the loss of the dystrophin glycoprotein complex.^19,20,21The α7β1 integrin is a heterodimeric laminin receptor involved in bidirectional cell signaling and is localized at junctional and extrajunctional sites in skeletal muscle.^22,23 At least six α7 integrin isoforms produced by developmentally regulated RNA splicing are expressed in skeletal muscle.²⁴ Mutations in the α7 integrin gene (ITGA7) cause myopathy in humans.²⁵ Mice lacking the α7 integrin develop myopathy, exhibit vascular smooth muscle defects and have altered extracellular matrix deposition.^{26,27,28,29,30} The observation that the α7β1 integrin is elevated in the muscle of DMD patients and mdx mice led to the hypothesis that the α7β1 integrin may compensate for the loss of dystrophin.¹⁹ Enhanced expression of the α7 integrin in the skeletal muscle of severely dystrophic mice reduced muscle pathology and increased lifespan by threefold.^10,11 In contrast, loss of both dystrophin and α7 integrin in mice results in severe muscular dystrophy and premature death by 4 weeks of age.^28,31 The α7β1 integrin is therefore a major modifier of disease progression in DMD.The utrophin glycoprotein complex is a third major laminin receptor in skeletal muscle. Utrophin has significant sequence homology to dystrophin.^32,33 In normal adult muscle utrophin is restricted to neuromuscular and myotendinous junctions.³⁴ During development or in damaged or diseased muscle, utrophin expression is increased and becomes localized at extrajunctional sites.^35,36 Utrophin interacts with the same proteins as dystrophin, but binds to actin filaments at different sites.³⁷ In mice, loss of utrophin results in a mild form of myasthenia with reduced sarcolemmal folding at the postsynaptic membrane of the neuromuscular junction.^12,15 Transgenic overexpression of utrophin has been shown to rescue mdx mice.³⁸ Mice that lack both dystrophin and utrophin exhibit severe muscular dystrophy and die by 14 weeks of age.^13,14 Thus, utrophin is also a major laminin receptor that modifies disease progression in DMD.To understand the functional overlap between the α7β1 integrin and utrophin in skeletal muscle, we produced mice that lack both α7 integrin and utrophin (α7/utr^−/−). Since both complexes are highly enriched at the MTJ and NMJ, we hypothesized that α7/utr^−/− mice may have severe abnormalities at these critical junctional sites. Our study demonstrates α7/utr^−/− mice exhibit partial embryonic lethality comparable with that observed in α7^−/− mice. Dystrophin is increased in these animals and enriched at the NMJ but not the MTJ. α7/utr^−/− mice display ultrastructural defects in their MTJ and compromised force transmission. Together, these results indicate that the α7β1 integrin, dystrophin and utrophin laminin binding complexes provide continuity between laminin in the extracellular matrix and the cell cytoskeleton, which are necessary for the normal structural and functional properties of skeletal muscle.     

Expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection: a role for osteopontin

To examine the roles of cytokines in muscle regeneration, we injected cardiotoxin into mouse tibialis anterior muscle and examined the expression profiles of cytokines and related genes in the regeneration process. Expression of 40, 64, and 7 genes among 522 genes spotted on a cytokine expression array were increased more than fivefold at 48 hours, 96 hours, and 7 days after toxin injection, respectively, when compared with those of the control muscle. Especially the levels of mRNA for chemokines and chemokine receptors, many of which are potent regulators of macrophages, were highly elevated 48 hours after injury. The expression of osteopontin (OPN), a versatile regulator of inflammation and tissue repair, was up-regulated more than 118-fold in regenerating muscle at 48 hours after injury. Northern blotting confirmed that the expression of OPN was highest at 48 hours after cardiotoxin injection and declined sharply thereafter. Immunohistochemistry showed that OPN was detected both in the cytoplasm of macrophages and in necrotic muscle infiltrated with macrophages. Our studies suggest OPN may serve as an adhesion molecule that promotes macrophage binding to necrotic fibers and may be an important mediator in the early phase of muscle regeneration.     

VCAM-1/α4β1 Integrin Interaction Is Crucial for Prompt Recruitment of Immune T Cells into the Brain during the Early Stage of Reactivation of Chronic Infection with Toxoplasma gondii To Prevent Toxoplasmic Encephalitis

Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4β1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8β, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4⁺ and CD8⁺ T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.     

Regulation of the atheroma-enriched protein, SPRR3, in vascular smooth muscle cells through cyclic strain is dependent on integrin alpha1beta1/collagen interaction

Atherosclerotic plaques express high levels of small proline-rich repeat protein (SPRR3), a previously characterized component of the cornified cell envelope of stratified epithelia, where it is believed to play a role in cellular adaptation to biomechanical stress. We investigated the physiological signals and underlying mechanism(s) that regulate atheroma-enriched SPRR3 expression in vascular smooth muscle cells (VSMCs). We showed that SPRR3 is expressed by VSMCs in both human and mouse atheromas. In cultured arterial VSMCs, mechanical cyclic strain, but neither shear stress nor lipid loading induced SPRR3 expression. Furthermore, this upregulation of SPRR3 expression was dependent on VSMC adherence to type I collagen. To link the mechanoregulation of SPRR3 to specific collagen/integrin interactions, we used blocking antibodies against either integrin α1 or α2 subunits and VSMCs from mice that lack specific collagen receptors. Our results showed a dependence on the α1β1 integrin for SPRR3 expression induced by cyclic strain. Furthermore, we showed that integrin α1 but not α2 subunits were expressed on VSMCs within mouse lesions but not in normal arteries. Therefore, we identified the enrichment of the mechanical strain-regulated protein SPRR3 in VSMCs of both human and mouse atherosclerotic lesions whose expression is dependent on the collagen-binding integrin α1β1 on VSMCs. These data suggest that SPRR3 may play a role in VSMC adaptation to local biomechanical stress within the plaque microenvironment.     

Laminin-111 protein therapy reduces muscle pathology and improves viability of a mouse model of merosin-deficient congenital muscular dystrophy

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a lethal muscle-wasting disease that is caused by mutations in the LAMA2 gene, resulting in the loss of laminin-α2 protein. MDC1A patients exhibit severe muscle weakness from birth, are confined to a wheelchair, require ventilator assistance, and have reduced life expectancy. There are currently no effective treatments or cures for MDC1A. Laminin-α2 is required for the formation of heterotrimeric laminin-211 (ie, α2, β1, and γ1) and laminin-221 (ie, α2, β2, and γ1), which are major constituents of skeletal muscle basal lamina. Laminin-111 (ie, α1, β1, and γ1) is the predominant laminin isoform in embryonic skeletal muscle and supports normal skeletal muscle development in laminin-α2-deficient muscle but is absent from adult skeletal muscle. In this study, we determined whether treatment with Engelbreth-Holm-Swarm-derived mouse laminin-111 protein could rescue MDC1A in the dy(W-/-) mouse model. We demonstrate that laminin-111 protein systemically delivered to the muscles of laminin-α2-deficient mice prevents muscle pathology, improves muscle strength, and dramatically increases life expectancy. Laminin-111 also prevented apoptosis in laminin-α2-deficient mouse muscle and primary human MDC1A myogenic cells, which indicates a conserved mechanism of action and cross-reactivity between species. Our results demonstrate that laminin-111 can serve as an effective protein substitution therapy for the treatment of muscular dystrophy in the dy(W-/-) mouse model and establish the potential for its use in the treatment of MDC1A.     

Knockout and knockin of the β1 exon D define distinct roles for integrin splice variants in heart function and embryonic development

The β1D integrin is a recently characterized isoform of the β1 subunit that is specifically expressed in heart and skeletal muscle. In this study we have assessed the function of the β1D integrin splice variant in mice by generating, for the first time, Cre-mediated exon-specific knockout and knockin strains for this splice variant. We show that removal of the exon for β1D leads to a mildly disturbed heart phenotype, whereas replacement of β1A by β1D results in embryonic lethality with a plethora of developmental defects, in part caused by the abnormal migration of neuroepithelial cells. Our data demonstrate that the splice variants A and D are not functionally equivalent. We propose that β1D is less efficient than β1A in mediating the signaling that regulates cell motility and responses of the cells to mechanical stress.     

Laminin α1 Regulates Age-Related Mesangial Cell Proliferation and Mesangial Matrix Accumulation through the TGF-β Pathway

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1^CKO) to study the role of LAMA1 in kidney development and function. Adult Lama1^CKO mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1^CKO mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-β1–induced Smad2 phosphorylation, and inhibitors of TGF-β1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1^CKO mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-β1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-β/Smad signaling.Laminins comprise a family of heterotrimeric extracellular matrix (ECM) proteins consisting of α, β, and γ chains^1,2 that regulate cell attachment, proliferation, and differentiation.^3,4 During early embryogenesis, laminin α1 (LAMA1) first appears at the 16-cell stage and is later present in the two basement membranes (BMs) formed before gastrulation, the embryonic BM, and Reichert’s membrane. In the developing kidney, temporal changes in laminin isoform expression occur as formation of glomeruli, the filtering unit of the kidney, proceeds. The earliest precursor of the glomerular BM (GBM) contains laminin-111 (LM-111; α1β1γ1). In contrast, the GBM contains LM-521 (α5β2γ1) at later developmental stages and in adulthood. Although LAMA1 is absent from the mature GBM, it is present in the glomerular mesangial matrix, an amorphous matrix made by mesangial cells that is one of the few prominent sites in which laminins are present outside of a definitive BM.⁵ The glomerular capillary wall consists of podocytes with interdigitated foot processes bridged by slit diaphragms, glomerular endothelial cells, and the intervening GBM⁶ that these two cell types together produce.⁷ Mesangial cells (MCs), the third cell type of the glomerulus, comprise approximately one-third of the glomerular tuft cell population. MCs bind the GBM at the base of the glomerular capillary loops to establish and maintain the structural architecture of the glomerular capillaries,⁶ similar to the function of certain microvascular pericytes. These cells also contribute to mesangial matrix homeostasis, regulate filtration surface area and capillary blood pressure, and phagocytose apoptotic cells and immune complexes formed at or delivered to the glomerular capillaries. Cell biological and biochemical studies have characterized MC responses to hormones, cytokines, growth factors, and metabolic, inflammatory, and immune mediators that are highly relevant to primary glomerular diseases or to systemic diseases that target glomerular cells.⁸Because inactivation of the Lama1 gene in mice results in a failure of assembly of Reichert’s membrane and developmental arrest shortly after implantation,⁹ in vivo studies of LAMA1 function have been limited. Herein, we used a conditional Lama1 knockout (KO) mouse model (Lama1^CKO) with specific deletion of Lama1 in the epiblast lineage using Sox2-Cre to study the role of LAMA1 in kidney development and function. We found that the absence of LAMA1 delayed glomerular development, and adult Lama1^CKO mice developed focal glomerulosclerosis and proteinuria with age. MCs from Lama1^CKO mice showed increased proliferation, resulting in expansion of the mesangial cell compartment. Thus, LAMA1 plays a critical role in MC homeostasis and kidney function.     

Administration of granulocyte colony-stimulating factor facilitates the regenerative process of injured mice skeletal muscle via the activation of Akt/GSK3αβ signals

Effects of administration of granulocyte colony-stimulating factor (G-CSF) on the regeneration of injured mammalian skeletal muscles were studied in male C57BL/6J mice. Muscle injury was induced by injection of cardiotoxin (CTX) into tibialis anterior muscles bilaterally. G-CSF was administrated for 8 consecutive days from 3 days before and 5 days after the injection. Significant decreases of wet weight and protein content were noted in the necrotic muscle with CTX injection. A large number of the regenerating fibers having central nucleus were observed 7 days after the injection. The regeneration of injured muscle was further facilitated by the G-CSF treatment. Population of Pax7-positive nuclei was increased by the G-CSF treatment at day 7. Phospho-Akt and phospho-glycogen synthase kinase 3αβ (GSK3αβ) signals were also activated by G-CSF-administrated group during the regenerative process. It was suggested that G-CSF treatment may facilitate the regeneration of injured skeletal muscles via the activation of Akt/GSK3αβ signals.     

Missense mutation of c.635 T > C in CAPN3 impairs muscle injury repair in a Limb-Girdel Muscular Dystropy Model

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a specific LGMD caused by a gene mutation encoding the calcium-dependent neutral cysteine protease calpain-3 (CAPN3). In our study, the compound heterozygosity with two missense variants c.635 T > C (p.Leu212Pro) and c.2120A > G (p.Asp707Gly) was identified in patients with LGMDR1. However, the pathogenicity of c.635 T > C has not been investigated. To evaluate the effects of this novel likely pathogenic variant to the motor system, the mouse model with c.635 T > C variant was prepared by CRISPR/Cas9 gene editing technique. The pathological results revealed that a limited number of inflammatory cells infiltrated the endomyocytes of certain c.635 T > C homozygous mice at 10 months of age. Compared with wild-type mice, motor function was not significantly impaired in Capn3 c. 635 T > C homozygous mice. Western blot and immunofluorescence assays further indicated that the expression levels of the Capn3 protein in muscle tissues of homozygous mice were similar to those of wild-type mice. However, the arrangement and ultrastructural alterations of the mitochondria in the muscular tissues of homozygous mice were confirmed by electron microscopy. Subsequently, muscle regeneration of LGMDR1 was simulated using cardiotoxin (CTX) to induce muscle necrosis and regeneration to trigger the injury modification process. The repair of the homozygous mice was significantly worse than that of the control mice at day 15 and day 21 following treatment, the c.635 T > C variant of Capn3 exhibited a significant effect on muscle regeneration of homozygous mice and induced mitochondrial damage. RNA-sequencing results demonstrated that the expression levels of the mitochondrial-related functional genes were significantly downregulated in the mutant mice. Taken together, the results of the present study strongly suggested that the LGMDR1 mouse model with a novel c.635 T > C variant in the Capn3 gene was significantly dysfunctional in muscle injury repair via impairment of the mitochondrial function.     

Cloning and characterization of developmental endothelial locus-1: An embryonic endothelial cell protein that binds the αvβ3 integrin receptor

We have taken advantage of an enhancer trap event in a line of transgenic mice to identify a unique developmentally regulated endothelial cell locus (Del1). The protein encoded in this locus contains three EGF-like repeats homologous to those in Notch and related proteins, including an EGF-like repeat that contains an RGD motif, and two discoidin I-like domains. Del1 is shown to be a matrix protein and to promote adhesion of endothelial cells through interaction with the αvβ3 integrin receptor. Embryonic endothelial-like yolk sac cells expressing recombinant Del1 protein, or grown on an extracellular matrix containing Del1 protein, are inhibited from forming vascular-like structures. Expression of Del1 protein in the chick chorioallantoic membrane leads to loss of vascular integrity and promotes vessel remodeling. Del1 is thus a new ligand for the αvβ3 integrin receptor and may function to regulate vascular morphogenesis or remodeling in embryonic development.     

Integrin α7 Binds Tissue Inhibitor of Metalloproteinase 3 to Suppress Growth of Prostate Cancer Cells

Integrin α7 (ITGA7) is a tumor-suppressor gene that is critical for suppressing the growth of malignant tumors; however, the mechanisms allowing ITGA7 to suppress the growth of cancer cells remain unclear. Herein, we show that ITGA7 binds to tissue inhibitor of metalloproteinase 3 (TIMP3) in prostate cancer cells. The ITGA7-TIMP3 binding led to a decreased protein level of tumor necrosis factor α, cytoplasmic translocation of NF-κB, and down-regulation of cyclin D1. These changes led to an accumulation of cells in G₀/G₁ and a dramatic suppression of cell growth. Knocking down TIMP3 or ITGA7/TIMP3 binding interference largely abrogated the signaling changes induced by ITGA7, whereas a mutant ITGA7 lacking TIMP3 binding activity had no tumor-suppressor activity. Interestingly, knocking down ITGA7 ligand laminin β1 enhanced ITGA7-TIMP3 signaling and the downstream tumor-suppressor activity, suggesting the existence of a counterbalancing role between extracellular matrix and integrin signaling. As a result, this report demonstrates a novel and critical signaling mechanism of ITGA7, through the TIMP3/NF-κB/cyclin D1 pathway.Integrin α7 (ITGA7) is a member of the extracellular matrix binding proteins. As a major class of cell adhesion molecules in mammalian cells, integrins are involved in many cellular processes, including development, immune responses, leukocyte trafficking, and hemostasis.¹ The integrin superfamily consists of 24 members, each of which mediates a unique function in mammals. The regulation of integrin expression is critical for certain aspects of tissue differentiation and regeneration (eg, keratinocyte differentiation, hair follicle formation, and skeletal muscle development),^2–4 and abnormal integrin expression is associated with several human diseases (eg, muscular dystrophy, Glanzmann’s thrombasthenia, and congenital cardiac myopathy).^4–6ITGA7 forms a heterodimer with integrin β1 in the plasma membrane and is responsible for communication between the extracellular matrix and cells.⁷ Itga7-deficient mice display significant hyperplasia and hypertrophy of arteries and arterioles and a malformation of skeletal muscles.^4,8 Recent mutational analysis revealed ITGA7 mutations in prostate cancer, hepatocellular carcinoma, soft tissue leiomyosarcoma, and glioblastoma multiforme, with frequencies ranging from 25% to 83%.⁹ Many of these mutations resulted in truncation, microdeletion, or frameshift of the protein. Interestingly, patients with prostate cancer or hepatocellular carcinoma harboring ITGA7 mutations also had a higher rate of clinical relapse.A meta-analysis of previously published microarray data^10–15 indicated that ITGA7 was down-regulated in nonmetastatic prostate cancer and leiomyosarcoma, but the magnitude of the down-regulation was larger in metastatic cancers. Also, prostate cancer and soft tissue leiomyosarcoma, with focal or no ITGA7 expression, were associated with a shorter metastasis-free survival time. The forced expression of normal ITGA7 in prostate cancer and leiomyosarcoma cell lines suppressed tumor growth and cancer cell migration in vitro. A mouse model of PC3 and DU145 xenograft prostate tumors showed a dramatic reduction in tumor volume, metastatic rate, and mortality rate when ITGA7 expression was restored. However, the molecular mechanism of ITGA7-mediated tumor-suppressor activity remains unclear. Herein, we report that the tissue inhibitor of metalloproteinase 3 (TIMP3), a matrix proteinase and a tumor suppressor, interacts with the C-terminus of ITGA7. We further show that the activation of ITGA7 leads to redistribution of NF-κB to the cytoplasm, the down-regulation of cyclin D1, and cell growth arrest.     

BMP-7 attenuates liver fibrosis via regulation of epidermal growth factor receptor

The aim of this study was to elucidate the effect of bone morphogenetic protein-7 (BMP-7) on liver fibrosis induced by carbon tetrachloride (CCl4) in vivo and on the hepatic stellate cells (HSC) activation in vitro. In vivo, thirty male ICR mice were randomly allocated to three groups, the control group (n = 6), the CCl4 group (n = 18) and the BMP-7+CCl4 group (n = 6). The model of liver fibrosis was induced by intraperitoneal injection with CCl4 three times per week lasting for 12 weeks in CCl4 group and the BMP-7+CCl4 group. After 8 weeks injection with CCl4, mice were intraperitoneal injected with human recombinant BMP-7 in BMP-7+CCl4 group. Meanwhile, mice in the CCl4 group were only intraperitoneal injection with equal amount of saline. The degree of liver fibrosis was assessed by HE and Masson’s staining. PCR and western blot were used to detect mRNA and protein levels. In BMP-7+CCl4 group, serum levels of alanine aminotransferase (ALT) and aminotransferase (AST) were decreased and serum albumin (Alb) was increased. Meanwhile, the expressions of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) were down-regulated by BMP-7 intervention as compared to the CCl4 group (P < 0.05). Furthermore, BMP-7 also suppressed the expression of epidermal growth factor receptor (EGFR) and phosphorylated-epidermal growth factor receptor (pEGFR). HE and Masson stain showed that liver damage was alleviated in BMP-7+CCl4 group. In vitro study, expression of EGFR, TGF-β1 and α-SMA were down regulated by BMP-7 dose-dependently, indicating it might effect on suppression of HSC activation. Therefore, our data indicate BMP-7 was capable of inhibiting liver fibrosis and suppressing HSCs activation, and these effects might rely on its crosstalk with EGFR and TGF-β1. We suggest that BMP-7 may be a potential reagentfor the prevention and treatment of liver fibrosis.     

Susceptibility to Salmonella typhimurium Infection and Effectiveness of Vaccination in Mice Deficient in the Tumor Necrosis Factor Alpha p55 Receptor

Mice defective in the ability to produce the tumor necrosis factor alpha p55 receptor (TNFαp55R) were orally challenged with a number of Salmonella typhimurium HWSH derivatives that differ in virulence. In comparison to TNFαp55R^+/+ mice, TNFαp55R^−/− mice succumbed earlier to challenge with wild-type S. typhimurium HWSH and S. typhimurium HWSH purE. In contrast, TNFαp55R^−/− mice were able to control an S. typhimurium HWSH aroA challenge, although greater numbers of Salmonella organisms were present in the tissues for a longer time period than was observed with TNFαp55R^+/+ mice. Vaccination of normal and TNFαp55R knockout animals with S. typhimurium HWSH aroA showed that TNFαp55R^−/− mice, unlike TNFαp55R^+/+ mice, were not protected against a virulent S. typhimurium HWSH challenge. Splenocytes from TNFαp55R^−/− mice exhibited a reduced ability to proliferate in the presence of S. typhimurium antigen compared to TNFαp55R^+/+ mice. Thus, TNFαp55R is essential for controlling Salmonella growth in tissues and for recall of immunity in murine salmonellosis.     

Homing Receptor α4β7 Integrin Expression Predicts Digestive Tract Involvement in Mantle Cell Lymphoma

Appropriate staging and evaluation of residual disease is critical to improving the treatment of patients with lymphoma. The specific expression of homing receptors may determine the preferential dissemination pattern of tumoral cells. We investigated the expression of the mucosal homing receptor α4β7 on tumoral cells from peripheral lymph node in patients with newly diagnosed mantle cell lymphoma (MCL) to check whether it is associated with gastrointestinal involvement. Expression of the α4β1 integrin and the peripheral lymph node addressin CD62L were also examined. Thirteen MCL patients presenting with peripheral lymphadenopathy were studied. Expression of the mucosal homing receptor integrin α4β7 by peripheral lymph node lymphoma cells was found to be frequent (5/13) and associated with gastrointestinal involvement (5/7). In contrast, lymphoma cells from patients without gastrointestinal involvement did not express α4β7 (6/6) (P = 0.03). These data suggest that α4β7 integrin is expressed by a subset of MCLs and that its expression may predict digestive tract involvement in MCL, furnishing a basis for recognizing two distinct clinical and phenotypic forms, ie, “digestive homing (or digestive primitive)” versus “peripheral” MCL. Further studies on more patients will be needed to understand the impact of biological differences on the prognosis of these two clinical forms.     

α1β1 Integrin/Rac1-Dependent Mesangial Invasion of Glomerular Capillaries in Alport Syndrome

Alport syndrome, hereditary glomerulonephritis with hearing loss, results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. The mechanism for delayed glomerular disease onset is unknown. Comparative analysis of Alport mice and CD151 knockout mice revealed progressive accumulation of laminin 211 in the glomerular basement membrane. We show mesangial processes invading the capillary loops of both models as well as in human Alport glomeruli, as the likely source of this laminin. l-NAME salt–induced hypertension accelerated mesangial cell process invasion. Cultured mesangial cells showed reduced migratory potential when treated with either integrin-linked kinase inhibitor or Rac1 inhibitor, or by deletion of integrin α1. Treatment of Alport mice with Rac1 inhibitor or deletion of integrin α1 reduced mesangial cell process invasion of the glomerular capillary tuft. Laminin α2–deficient Alport mice show reduced mesangial process invasion, and cultured laminin α2–null cells showed reduced migratory potential, indicating a functional role for mesangial laminins in progression of Alport glomerular pathogenesis. Collectively, these findings predict a role for biomechanical insult in the induction of integrin α1β1–dependent Rac1-mediated mesangial cell process invasion of the glomerular capillary tuft as an initiation mechanism of Alport glomerular pathology.Alport syndrome is characterized by delayed-onset progressive glomerulonephritis associated with sensorineural hearing loss and retinal flecks.¹ The most common form (80%) is X-linked and caused by mutations in the type IV collagen COL4A5 gene.² The two autosomal forms of the disease account for the remaining 20% of Alport patients, and result from mutations in the COL4A3 and COL4A4 genes.³ The α3(IV), α4(IV), and α5(IV) proteins form a heterotrimer that is assembled into a subepithelial network in the glomerular basement membrane (GBM) that is physically and biochemically distinct from a subendothelial type IV collagen network comprising α1(IV) and α2(IV) heterotrimers.⁴ Mutations in any one of the three type IV collagen genes that cause Alport syndrome result in the absence of all three proteins in the GBM due to an obligatory association to form functional heterotrimers.⁵ Thus, the net result for all genetic forms of Alport syndrome is the absence of the α3(IV) α4(IV) α5(IV) subepithelial collagen network, resulting in a GBM type IV collagen network comprising only α1(IV) and α2(IV) heterotrimers.This change in basement membrane composition does not result in immediate pathology. The GBM appears to function adequately for the first few years of life and sometimes past the first decade.⁶ This delayed onset predicts a triggering mechanism for glomerular disease initiation and a theoretical window for therapeutic intervention that may arrest or significantly ameliorate Alport renal disease in its earliest stages. The activation of genes encoding GBM matrix molecules, matrix metalloproteinases (MMPs), and proinflammatory cytokines have all been linked to the progression of Alport glomerular disease. These, however, are events that occur after the onset of proteinuria, and therefore, downstream of disease initiation events.^7–11 Consistent with this notion, experiments aimed at blocking these pathways have offered only limited therapeutic benefit in mouse models for Alport syndrome.^8–10,12 One of the earliest events we have documented is the appearance of an irregular deposition of laminin 211 in the GBM of Alport mice,⁸ an observation confirmed in both Alport dogs and human patients with the disease.¹³ This laminin is normally found only in the mesangium of the glomerulus, and is not expressed in the GBM at any stage of embryonic development.¹⁴ Indeed, several other mesangial matrix proteins appear in the GBM of Alport mice, including laminin 111 and fibronectin.^15,16In the Alport glomerulus, the podocytes are exposed to GBM that has an embryonic type IV collagen composition.^17,18 This could result in altered cell signaling that may trigger the onset of the disease. It has been proposed that this type of mechanism may account for the reactivation of laminin 111 expression in podocytes,¹⁹ because laminin 111 is found in the GBM during development.¹⁴ Because the α1(IV)/α2(IV) collagen network contains significantly fewer interchain disulfide crosslinks,²⁰ and the Alport GBM is thinner than normal,²¹ the Alport GBM is likely to be more elastic, resulting in elevated biomechanical strain on the glomerular cells at their points of contact with the GBM. Consistently, glomeruli from Alport mice have been shown to have elevated deformability relative to wild-type glomeruli,²² and salt-induced hypertension has been shown to accelerate glomerular disease progression in Alport mice.²³In this work, we show that the cellular origin of GBM laminin 211 in Alport glomeruli is mesangial cell process invasion, and that deletion of laminin 211 in Alport mice ameliorates the mesangial process invasion of the glomerular capillary loops in Alport mice. Salt-mediated hypertension exacerbates this mesangial process invasion. A knockout mouse for the integrin α3β1 coreceptor CD151 also develops mesangial process invasion of the capillary loops with GBM deposition of laminin 211, demonstrating the same phenotype for a completely unrelated component of the capillary structural barrier. The CD151 knockout mouse model also shows accelerated glomerular disease progression in response to hypertension.²⁴ We show that biomechanical stretching of cultured mesangial cells induces promigratory cytokines transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF), both known to be induced in Alport glomeruli.^7,12 Inhibitor studies indicate that mesangial cell migration is mediated by integrin α1β1 signaling through the Rho GTPases RAC1 and CDC42. Consistently, integrin α1 deletion in Alport mice was previously shown to ameliorate glomerular disease progression and slow the accumulation of laminin 211 in Alport GBM.⁸ Here, we show that mesangial process invasion of the capillary loops is ameliorated in integrin α1–null Alport mice. These data define a role for biomechanical strain-mediated induction of mesangial cell process invasion as a key aspect of Alport glomerular disease initiation, and set the stage for defining novel therapeutic targets aimed at blocking this process.