Protein post-translational modifications like glycation, carbamylation and citrullination increase the functional diversity of the proteome but in disease situations might do more harm than good. Post-translational modifications of ECM proteins are thus appearing as mechanisms, which contribute to tissue dysfunction in chronic kidney disease, in diabetes and in various inflammatory diseases. In chronic renal failure, carbamylation could lead to kidney fibrosis. In diabetes, high glucose levels lead to non-enzymatic glycation and cross-linking of collagens, which contribute to tissue stiffening with consequences for cardiovascular and renal functions. In inflammatory diseases, citrullination deiminates arginine residues with possible consequences for integrin-mediated cell adhesion to RGD- and GFOGER sequences in ECM proteins. Citrullination of fibronectin was in one study suggested to affect cell adhesion by modifying the heparin-binding site and not the RGD site. In a recent publication citrullination of GFOGER sequences in collagen II was demonstrated to selectively affect ?10?1 and ?11?1 integrin-mediated cell adhesion to collagen II, with consequences for synovial fibroblast and stem cell adhesion and migration. The implications of citrullination affecting integrin binding in disease open up a new area of study and might have implications for the pathogenesis of inflammatory diseases like rheumatoid arthritis and periodontitis.
Integrin ?11 is the last addition to the vertebrate integrin family. In this chapter we will summarize some basic facts about this integrin and update with information that has been gained in the last decade. Integrin ?11?1 is a major collagen receptor on a subset of fibroblasts. Extensive characterization of the expression pattern in developing mouse embryos has demonstrated expression restricted to subsets of fibroblasts and a transient expression in odontoblasts, but comprehensive characterization of corresponding expression in adult tissues is still lacking. Mice lacking integrin ?11 are dwarfed, primarily due to defective incisor eruption defect, which can be traced back to need for ?11 on periodontal ligament fibroblasts during incisor eruption. Separate studies have suggested reduced levels of IGF-1 in mice lacking ?11. Analysis of lung cancer has identified ?11?1 as a functional important collagen receptor on carcinoma associated fibroblasts (CAFs) and a number of disease models are awaiting analysis to see the importance of this collagen receptor in pathological models.
Collagen XXII, a FACIT (fibril-associated collagen with interrupted triple helices), is expressed at the myotendinous junction and the articular surface of joint cartilage. Cellular receptors like collagen-binding integrins are known to bind collagens with distinct binding motifs following the sequence GXOGER. In the present study, we demonstrate the sequences GLQGER and GFKGER as novel binding motifs between collagen XXII and collagen-binding integrins, especially ?2?1 integrin. Solid-phase assays and surface plasmon resonance spectroscopy revealed a direct interaction between ?2?1 integrin and the motif GFKGER. In addition, immunohistochemical analysis demonstrated partial co-localization of collagen XXII, ?2?1 integrin and ?11?1 integrin at the myotendinous junction. Furthermore, computational modelling of the motifs GLQGER and GFKGER showed perfect fitting of the sequences into the binding pocket of collagen-binding integrins. Taken together, we demonstrated that collagen XXII interacts with collagen-binding integrins via the new motifs GLQGER and GFKGER.
Laminins (LM), basement membrane molecules and mediators of epithelial-stromal communication, are crucial in tissue homeostasis. Inflammatory Bowel Diseases (IBD) are multifactorial pathologies where the microenvironment and in particular LM play an important yet poorly understood role in tissue maintenance, and in cancer progression which represents an inherent risk of IBD. Here we showed first that in human IBD colonic samples and in murine colitis the LM?1 and LM?5 chains are specifically and ectopically overexpressed with a concomitant nuclear p53 accumulation. Linked to this observation, we provided a mechanism showing that p53 induces LM?1 expression at the promoter level by ChIP analysis and this was confirmed by knockdown in cell transfection experiments. To mimic the human disease, we induced colitis and colitis-associated cancer by chemical treatment (DSS) combined or not with a carcinogen (AOM) in transgenic mice overexpressing LM?1 or LM?5 specifically in the intestine. We demonstrated that high LM?1 or LM?5 expression decreased susceptibility towards experimentally DSS-induced colon inflammation as assessed by histological scoring and decrease of pro-inflammatory cytokines. Yet in a pro-oncogenic context, we showed that LM would favor tumorigenesis as revealed by enhanced tumor lesion formation in both LM transgenic mice. Altogether, our results showed that nuclear p53 and associated overexpression of LM?1 and LM?5 protect tissue from inflammation. But in a mutation setting, the same LM molecules favor progression of IBD into colitis-associated cancer. Our transgenic mice represent attractive new models to acquire knowledge about the paradoxical effect of LM that mediate either tissue reparation or cancer according to the microenvironment. In the early phases of IBD, reinforcing basement membrane stability/organization could be a promising therapeutic approach.
We have previously shown that fibroblast expression of ?11?1 integrin stimulates A549 carcinoma cell growth in a xenograft tumor model. To understand the molecular mechanisms whereby a collagen receptor on fibroblast can regulate tumor growth we have used a 3D heterospheroid system composed of A549 tumor cells and fibroblasts without (?11+/+) or with a deletion (?11-/-) in integrin ?11 gene. Our data show that ?11-/-/A549 spheroids are larger than ?11+/+/A549 spheroids, and that A549 cell number, cell migration and cell invasion in a collagen I gel are decreased in ?11-/-/A549 spheroids. Gene expression profiling of differentially expressed genes in fibroblast/A549 spheroids identified CXCL5 as one molecule down-regulated in A549 cells in the absence of ?11 on the fibroblasts. Blocking CXCL5 function with the CXCR2 inhibitor SB225002 reduced cell proliferation and cell migration of A549 cells within spheroids, demonstrating that the fibroblast integrin ?11?1 in a 3D heterospheroid context affects carcinoma cell growth and invasion by stimulating autocrine secretion of CXCL5. We furthermore suggest that fibroblast ?11?1 in fibroblast/A549 spheroids regulates interstitial fluid pressure by compacting the collagen matrix, in turn implying a role for stromal collagen receptors in regulating tensional hemostasis in tumors. In summary, blocking stromal ?11?1 integrin function might thus be a stroma-targeted therapeutic strategy to increase the efficacy of chemotherapy.
Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit ?11 mRNA and protein expression, and an increase in ?2 integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-?1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced.
Despite detailed knowledge about the structure and signaling properties of individual collagen receptors, much remains to be learned about how these receptors participate in linking cells to fibrillar collagen matrices in tissues. In addition to collagen-binding integrins, a group of proteins with affinity both for fibrillar collagens and integrins link these two protein families together. We have introduced the name COLINBRI (COLlagen INtegrin BRIdging molecules) for this set of molecules. Whereas collagens are the major building blocks in tissues, defects in these structural proteins have severe consequences for tissue integrity; the mild phenotypes of the integrin type of collagen receptors have raised questions about their importance in tissue biology and pathology. Scope of review We will discuss the two types of cell linkages to fibrillar collagen (direct- versus indirect COLINBRI-mediated) and discuss how the parallel existence of direct and indirect linkages to collagens may ensure tissue integrity. Major conclusions The observed mild phenotypes of mice deficient in collagen-binding integrins and the relatively restricted availability of integrin-binding sequences in mature fibrillar collagens matrices support the existence of indirect collagen binding mechanisms in parallel with direct collagen binding in vivo. General significance A continued focus on understanding the molecular details of cell adhesion mechanisms to collagens will be important and will benefit our understanding of diseases like tissue- and tumor fibrosis where collagen dynamics are disturbed.
The periodontal ligament is the tissue that connects teeth to bone. The periodontal ligament is a fascinating tissue from a cell biologists point of view, and because of its special properties and stem-cell content it has also come into the limelight in emerging fields of regenerative medicine. An increased range of genetically modified mouse models offer new tools for studying molecular mechanisms of tooth development. However, owing to species-specific organization of the tooth apparatus, the use of genetic animal models to study the role of the periodontal ligament in normal human tooth physiology and tooth pathology is challenging.
We have previously determined that integrin ?11?1 is required on mouse periodontal ligament (PDL) fibroblasts to generate the force needed for incisor eruption. As part of the phenotype of ?11(-/-) mice, the incisor PDL (iPDL) is thickened, due to disturbed matrix remodeling. To determine the molecular mechanism behind the disturbed matrix dynamics in the PDL we crossed ?11(-/-) mice with the Immortomouse and isolated immortalized iPDL cells. Microarray analysis of iPDL cells cultured inside a 3D collagen gel demonstrated downregulated expression of a number of genes in ?11-deficient iPDL cells, including matrix metalloproteinase-13 (MMP-13) and cathepsin K. ?11(-/-) iPDL cells in vitro displayed disturbed interactions with collagen I during contraction of attached and floating collagen lattices and furthermore displayed reduced MMP-13 protein expression levels. The MMP-13 specific inhibitor WAY 170523 and the Cathepsin K Inhibitor II both blocked part of the ?11 integrin-mediated collagen remodeling. In summary, our data demonstrate that in iPDL fibroblasts the mechanical strain generated by ?11?1 integrin regulates molecules involved in collagen matrix dynamics. The positive regulation of ?11?1-dependent matrix remodeling, involving MMP-13 and cathepsin K, might also occur in other types of fibroblasts and be an important regulatory mechanism for coordinated extracellular and intracellular collagen turnover in tissue homeostasis.
Here we describe a NOD/Scid mouse strain expressing the dsRed transgene. The strain is maintained by inbreeding of homozygous dsRed NOD/Scid siblings, and expresses red fluorescence from various organs. The model allows engraftment of human tumor tissue, and engrafted tumors were separated into stromal and malignant cell compartments. Furthermore, we compared tumor-associated and normal fibroblast for expression of fibroblast-associated markers, and identified a marker panel that was upregulated in the tumor-associated fibroblasts. In conclusion, we propose that this model may be used in a variety of studies of tumor progression and to elucidate the role of the tumor microenvironment.
Mice with a combined deficiency in the ?2?1 and ?11?1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either ?2?1 or ?11?1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis.
Fibrotic tissue is characterized by an overabundance of myofibroblasts. Thus, understanding the factors that induce myofibroblast differentiation is paramount to preventing fibrotic healing. Previous studies have shown that mechanical stress derived from the integrin-mediated interaction between extracellular matrix and the cytoskeleton promotes myofibroblast differentiation. Integrin alpha11beta1 is a collagen receptor on fibroblasts. To determine whether alpha11beta1 can act as a mechanosensor to promote the myofibroblast phenotype, mouse embryonic fibroblasts and human corneal fibroblasts were utilized. We found that alpha11 mRNA and protein levels were up-regulated in mouse embryonic fibroblasts grown in attached three-dimensional collagen gels and conversely down-regulated in cells grown in floating gels. alpha11 up-regulation could be prevented by manually detaching the collagen gels or by cytochalasin D treatment. Furthermore, SB-431542, an inhibitor of signaling via ALK4, ALK5, and ALK7, prevented the up-regulation of alpha11 and the concomitant phosphorylation of Smad3 under attached conditions. In attached gels, TGF-beta1 was secreted in its inactive form but surprisingly not further activated, thus not influencing alpha11 regulation. However, inhibition of activin A attenuated the up-regulation of alpha11. To determine the role of alpha11 in myofibroblast differentiation, human corneal fibroblasts were transfected with small interfering RNA to alpha11, which decreased alpha-smooth muscle actin expression and myofibroblast differentiation. Our data suggest that alpha11beta1 is regulated by cell/matrix stress involving activin A and Smad3 and that alpha11beta1 regulates myofibroblast differentiation.
Endorepellin, the C-terminal domain of perlecan, is a powerful angiogenesis inhibitor. To dissect the mechanism of endorepellin-mediated endothelial silencing, we used an antibody array against multiple tyrosine kinase receptors. Endorepellin caused a widespread reduction in phosphorylation of key receptors involved in angiogenesis and a concurrent increase in phosphatase activity in endothelial cells and tumor xenografts. These effects were efficiently hampered by function-blocking antibodies against integrin alpha2beta1, the functional endorepellin receptor. The Src homology-2 protein phosphatase-1 (SHP-1) coprecipitated with integrin alpha2 and was phosphorylated in a dynamic fashion after endorepellin stimulation. Genetic evidence was provided by lack of an endorepellin-evoked phosphatase response in microvascular endothelial cells derived from integrin alpha2beta1(-/-) mice and by response to endorepellin in cells genetically engineered to express the alpha2beta1 integrin, but not in cells either lacking this receptor or expressing a chimera harboring the integrin alpha2 ectodomain fused to the alpha1 intracellular domain. siRNA-mediated knockdown of integrin alpha2 caused a dose-dependent reduction of SHP-1. Finally, the levels of SHP-1 and its enzymatic activity were substantially reduced in multiple organs from alpha2beta1(-/-) mice. Our results show that SHP-1 is an essential mediator of endorepellin activity and discover a novel functional interaction between the integrin alpha2 subunit and SHP-1.
Integrin alpha11beta1 is expressed by ectomesenchymally- and mesodermally-derived fibroblasts and is the major collagen receptor on embryonic fibroblasts. We have previously characterized a 3kb human alpha11 promoter region in vitro. In the current study we generated promoter-LacZ reporter transgenic mice to examine the ability of the 3kb alpha11 promoter to drive tissue-specific expression also in vivo. Our data show that the 3 kb alpha11 promoter contains most of the regulatory elements that direct ectomesenchymal and mesodermal fibroblast-specific expression. Not much is known about integrin alpha11 regulation by TGF-beta family members and the potential role of alpha11 in TGF-beta1 driven processes such as fibrosis and wound contraction. In the current study we show that TGF-beta1 induces alpha11 transcription in the fibrosarcoma cell line HT1080 as well as in primary fibroblasts. Co-transfection of an expression plasmid encoding constitutively active ALK5 together with alpha11 promoter-luciferase reporter constructs demonstrated that TGF-beta1 responsive elements are located within the 3kb alpha11 promoter. Serial deletions located TGF-beta1 responsiveness to the proximal promoter (nt -176/+25) as well as to the region extending to nt -330. Transfection and expression of the inhibitory Smad7 in the cells attenuated the TGF-beta1-dependent alpha11 induction both at the RNA and the protein level. Mutation and deletion analyses identified a Smad-binding element, SBE2 (nt -182/-176), as an important Smad3-binding site in this part of the promoter. Further analyses suggested that the Sp1-binding site SBS1 (nt -140/-134) takes part in the responsiveness to TGF-beta1 in a Smad2-dependent manner. In summary, our data confirm that 3kb of the alpha11 promoter is efficient in driving tissue-specific expression in vivo. We also demonstrate that this promoter confers TGF-beta1 responsiveness which appears to rely on both a Smad-binding element at nt -182/-176 and a Sp1-binding site at nt -140/-134. Our data furthermore indicate that additional elements needed for TGF-beta1 responsiveness are located upstream in the -2962/-330 promoter region.
Alpha-dystroglycan (alpha-DG) represents a highly glycosylated cell surface molecule that is expressed in the epithelial cell-basement membrane (BM) interface and plays an essential role in epithelium development and tissue organization. The alpha-DG-mediated epithelial cell-BM interaction is often impaired in invasive carcinomas, yet roles and underlying mechanisms of such an impaired interaction in tumor progression remain unclear. We report here a suppressor function of laminin-binding glycans on alpha-DG in tumor progression. In aggressive prostate and breast carcinoma cell lines, laminin-binding glycans are dramatically decreased, although the amount of alpha-DG and beta-dystroglycan is maintained. The decrease of laminin-binding glycans and consequent increased cell migration were associated with the decreased expression of beta3-N-acetylglucosaminyltransferase-1 (beta3GnT1). Forced expression of beta3GnT1 in aggressive cancer cells restored the laminin-binding glycans and decreased tumor formation. beta3GnT1 was found to be required for laminin-binding glycan synthesis through formation of a complex with LARGE, thus regulating the function of LARGE. Interaction of the laminin-binding glycans with laminin and other adhesive molecules in BM attenuates tumor cell migratory potential by antagonizing ERK/AKT phosphorylation induced by the components in the ECM. These results identify a previously undescribed role of carbohydrate-dependent cell-BM interaction in tumor suppression and its control by beta3GnT1 and LARGE.
Integrins are cell adhesion receptors that are evolutionary old and that play important roles during developmental and pathological processes. The integrin family is composed of 24 alphabeta heterodimeric members that mediate the attachment of cells to the extracellular matrix (ECM) but that also take part in specialized cell-cell interactions. Only a subset of integrins (8 out of 24) recognizes the RGD sequence in the native ligands. In some ECM molecules, such as collagen and certain laminin isoforms, the RGD sequences are exposed upon denaturation or proteolytic cleavage, allowing cells to bind these ligands by using RGD-binding receptors. Proteolytic cleavage of ECM proteins might also generate fragments with novel biological activity such as endostatin, tumstatin, and endorepellin. Nine integrin chains contain an alphaI domain, including the collagen-binding integrins alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1. The collagen-binding integrins recognize the triple-helical GFOGER sequence in the major collagens, but their ability to recognize these sequences in vivo is dependent on the fibrillar status and accessibility of the interactive domains in the fibrillar collagens. The current review summarizes some basic facts about the integrin family including a historical perspective, their structure, and their ligand-binding properties.
The integrins form a large family of cell adhesion receptors. All multicellular animals express integrins, indicating that the family evolved relatively early in the history of metazoans, and homologous sequences of the component domains of integrin alpha and beta subunits are seen in prokaryotes. Some integrins, however, seem to be much younger. For example, the alphaI domain containing integrins, including collagen receptors and leukocyte integrins, have been found in chordates only. Here, we will discuss what conclusions can be drawn about integrin function by studying the evolutionary conservation of integrins. We will also look at how studying integrins in organisms such as the fruit fly and mouse has helped our understanding of integrin evolution-function relationships. As an illustration of this, we will summarize the current understanding of integrin involvement in skeletal muscle formation.
Diabetic cardiomyopathy is characterized by the production of a disorganized fibrotic matrix in the absence of coronary atherosclerosis and hypertension. We examined whether adhesion of cardiac fibroblasts to glycated collagens mediates the differentiation of pro-fibrotic myofibroblasts, which may contribute to cardiac fibrosis.
Stromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model.
Integrins are well characterized cell surface receptors for extracellular matrix proteins. Mapping integrin-binding sites within the fibrillar collagens identified GFOGER as a high affinity site recognized by ?2?1, but with lower affinity for ?1?1. Here, to identify specific ligands for ?1?1, we examined binding of the recombinant human ?1 I domain, the rat pheochromocytoma cell line (PC12), and the rat glioma Rugli cell line to our collagen Toolkit II and III peptides using solid-phase and real-time label-free adhesion assays. We observed Mg(2+)-dependent binding of the ?1 I domain to the peptides in the following rank order: III-7 (GLOGEN), II-28 (GFOGER), II-7 and II-8 (GLOGER), II-18 (GAOGER), III-4 (GROGER). PC12 cells showed a similar profile. Using antibody blockade, we confirmed that binding of PC12 cells to peptide III-7 was mediated by integrin ?1?1. We also identified a new ?1?1-binding activity within peptide II-27. The sequence GVOGEA bound weakly to PC12 cells and strongly to activated Rugli cells or to an activated ?1 I domain, but not to the ?2 I domain or to C2C12 cells expressing ?2?1 or ?11?1. Thus, GVOGEA is specific for ?1?1. Although recognized by both ?2?1 and ?11?1, GLOGEN is a better ligand for ?1?1 compared with GFOGER. Finally, using biosensor assays, we show that although GLOGEN is able to compete for the ?1 I domain from collagen IV (IC(50) ?3 ?m), GFOGER is much less potent (IC(50) ?90 ?m), as shown previously. These data confirm the selectivity of GFOGER for ?2?1 and establish GLOGEN as a high affinity site for ?1?1.
Short (or small) interfering RNAs (siRNAs) are double-stranded RNA molecules about 21-25 nucleotides long that have the capacity to disrupt the activity of genes on a posttranscriptional level. This sequence homology-driven gene silencing capacity has been utilized by researchers to selectively block the translation of mRNA to proteins in order to study specific gene functions and identify target molecules. Importantly, siRNAs have the potential to be used in treatment of disease. Here, we describe how the siRNA technology can be used to knock down genes in dental tissue-derived cells using integrin ?11 knockdown as an example.
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