Cooperation between integrins and growth factor receptors in signaling and endocytosis.

All multicellular animals express receptors for growth factors (GFs) and extracellular matrix (ECM) molecules. Integrin-type ECM receptors anchor cells to their surroundings and concomitantly activate intracellular signal transduction pathways. The same signaling mechanisms are regulated by GF receptors (GFRs). Recently, intensive research efforts have revealed novel mechanisms describing how the two receptor systems collaborate at many different levels. Integrins can directly bind to GFs and promote their activation. Adhesion receptors also organize signaling platforms and assist GFRs or even activate them via ligand-independent mechanisms. Furthermore, integrins can orchestrate endocytosis and recycling of GFRs. Here, we review the present knowledge about the interplay between integrins and GFRs and discuss recent ideas of how this collaboration may explain some previous controversies in integrin research.

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

Integrin α11ß1 is a receptor for collagen XIII.

Collagen XIII is a conserved transmembrane collagen mainly expressed in mesenchymal tissues. Previously, we have shown that collagen XIII modulates tissue development and homeostasis. Integrins are a family of receptors that mediate signals from the environment into the cells and vice versa. Integrin α11ß1 is a collagen receptor known to recognize the GFOGER (O=hydroxyproline) sequence in collagens. Interestingly, collagen XIII and integrin α11ß1 both have a role in the regulation of bone homeostasis. To study whether α11ß1 is a receptor for collagen XIII, we utilized C2C12 cells transfected to express α11ß1 as their only collagen receptor. The interaction between collagen XIII and integrin α11ß1 was also confirmed by surface plasmon resonance and pull-down assays. We discovered that integrin α11ß1 mediates cell adhesion to two collagenous motifs, namely GPKGER and GF(S)QGEK, that were shown to act as the recognition sites for the integrin α11-I domain. Furthermore, we studied the in vivo significance of the α11ß1-collagen XIII interaction by crossbreeding α11 null mice (Itga11-/-) with mice overexpressing Col13a1 (Col13a1oe). When we evaluated the bone morphology by microcomputed tomography, Col13a1oe mice had a drastic bone overgrowth followed by severe osteoporosis, whereas the double mutant mouse line showed a much milder bone phenotype. To conclude, our data identifies integrin α11ß1 as a new collagen XIII receptor and demonstrates that this ligand-receptor pair has a role in the maintenance of bone homeostasis.

Interaction between prostate cancer cells and prostate fibroblasts promotes accumulation and proteolytic processing of basement membrane proteins.

BACKGROUND: Tumor microenvironment or stroma has the potency to regulate the behavior of malignant cells. Fibroblast-like cells are abundant in tumor stroma and they are also responsible for the synthesis of many extracellular matrix components. Fibroblast-cancer cell interplay can modify the functions of both cell types. METHODS: We applied mass spectrometry and proteomics to unveil the matrisome in 3D spheroids formed by DU145 prostate cancer cells, PC3 prostate cancer cells, or prostate-derived fibroblasts. Similarly, DU145/fibroblast and PC3/fibroblast coculture spheroids were also analyzed. Western blot analysis and immunofluorescence were used to confirm the presence of specific proteins in spheroids. Cancer dissemination was studied by utilizing "out of spheroids" migration and invasion assays. RESULTS: In the spheroid model cancer cell-fibroblast interplay caused remarkable changes in the extracellular matrix and accelerated the invasion of DU145 cells. Fibroblasts produced structural matrix proteins, growth factors, and matrix metalloproteinases. In cancer cell/fibroblast cocultures basement membrane components, including laminins (α3, α5, ß2, and ß3), heparan sulfate proteoglycan (HSPG2 gene product), and collagen XVIII accumulated in a prominent manner when compared with spheroids that contained fibroblasts or cancer cells only. Furthermore, collagen XVIII was intensively processed to different endostatin-containing isoforms by cancer cell-derived cathepsin L. CONCLUSIONS: Fibroblasts can promote carcinoma cell dissemination by several different mechanisms. Extracellular matrix and basement membrane proteins provide attachment sites for cell locomotion promoting adhesion receptors. Growth factors and metalloproteinases are known to accelerate cell invasion. In addition, cancer cell-fibroblast interplay generates biologically active fragments of basement membrane proteins, such as endostatin.

Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms.

Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC-MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1ß1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix.

The composition of prostate core matrisome in vivo and in vitro unveiled by mass spectrometric analysis.

BACKGROUND: The composition and organization of extracellular matrix (ECM) are important regulators of cell behavior. In particular in the prostate, this central role of the ECM is further stressed by the fact that several potential markers of prostate stem cells are matrix receptors. METHODS: We established 12 fibroblastic cell lines from cancerous and non-cancerous areas of six prostates and allowed the cells to produce ECM under cell culture conditions. We also performed a proteome wide analysis of the ECM components by mass spectrometry. To study the in vitro activation of fibroblastic cells we compared the differences between the ECM produced in cell culture by six non-cancerous-tissue-derived fibroblasts and the in vivo matrisome from the corresponding non-cancerous tissue of prostate. RESULTS: Our results suggest that at tissue level the ECM is mainly produced by fibroblastic cells and that it contains standard collagen I fibrils and fibril-associated proteins. Beaded-filament forming collagen VI is also abundant and basement membranes potentially contain five laminin subtypes and collagens XV and XVIII. As the main finding, we also detected differences when in vivo and in vitro matrisomes were compared. Only 65 out of 206 proteins were found to be common for both in vivo and in vitro samples. Majority of the 55 proteins, which were solely detected in in vivo samples, were considered to be plasma derived. Eighty-six proteins were solely found from in vitro fibroblast-derived ECM, and most of them were related to matrix remodeling or growth factor action, proposing that the activation of fibroblasts in cell culture may remarkably modify their gene expression profile. Finally, in comparison to traditional 2D in vitro cell culture, the ECM composition of 3D spheroid culture was analyzed. The matrisome in spheroid culture did not resemble the in vivo ECM more closely than in monolayer culture. CONCLUSIONS: Artificial activation of ECM remodeling seems to be a distinctive feature in in vitro models. In conclusion the constitution of ECM produced by prostate derived fibroblasts in vitro is similar, but not identical to the prostate ECM in vivo as shown here by mass spectrometric analysis.

The PIM1 kinase promotes prostate cancer cell migration and adhesion via multiple signalling pathways.

The ability of cells to migrate and form metastases is one of the fatal hallmarks of cancer that can be conquered only with better understanding of the molecules and regulatory mechanisms involved. The oncogenic PIM kinases have been shown to support cancer cell survival and motility, but the PIM-regulated pathways stimulating cell migration and invasion are less well characterized than those affecting cell survival. Here we have identified the glycogen synthase kinase 3ß (GSK3B) and the forkhead box P3 (FOXP3) transcription factor as direct PIM targets, whose tumour-suppressive effects in prostate cancer cells are inhibited by PIM-induced phosphorylation, resulting in increased cell migration. Targeting GSK3B is also essential for the observed PIM-enhanced expression of the prostaglandin-endoperoxide synthase 2 (PTGS2), which is an important regulator of both cell migration and adhesion. Accordingly, selective inhibition of PIM activity not only reduces cell migration, but also affects integrin-mediated cell adhesion. Taken together, these data provide novel mechanistic insights on how and why patients with metastatic prostate cancer may benefit from therapies targeting PIM kinases, and how such approaches may also be applicable to inflammatory conditions.

Platelet response to a small molecule inhibitor of α2ß1 integrin is associated with ITGA2 C807T dimorphism.

High expression of the collagen receptor, α2ß1 integrin, on platelets of ITGA2 807T-allele carriers has been identified as a risk factor for thromboembolic conditions, and α2ß1 inhibitors are considered to be potential therapeutic agents. In 59 genotyped individuals, we measured α2 expression levels on platelets and analyzed platelet adhesion to collagen under flow conditions. A sulfonamide-type small-molecule inhibitor of α2ß1 integrin decreased average platelet adhesion in individuals with the C/T807T genotype but not in those harboring C807C. Thus, genotype can be used to select a human subpopulation that has the highest probability of showing a positive response to α2ß1 inhibitors.

Critical role for αvß6 integrin in enamel biomineralization.

Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with the ameloblast plasma membrane. Although it is known that integrins mediate cell-matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. We hypothesized that αvß6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both ß6 integrin mRNA and protein. The maxillary incisors of Itgb6(-/-) mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6(-/-) mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6(-/-) enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6(-/-) mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, in which αvß6 integrin was not an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin αvß6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition and/or turnover and subsequent enamel biomineralization.

BioImageXD: an open, general-purpose and high-throughput image-processing platform.

BioImageXD puts open-source computer science tools for three-dimensional visualization and analysis into the hands of all researchers, through a user-friendly graphical interface tuned to the needs of biologists. BioImageXD has no restrictive licenses or undisclosed algorithms and enables publication of precise, reproducible and modifiable workflows. It allows simple construction of processing pipelines and should enable biologists to perform challenging analyses of complex processes. We demonstrate its performance in a study of integrin clustering in response to selected inhibitors.

Citrullination of collagen II affects integrin-mediated cell adhesion in a receptor-specific manner.

Citrullinated collagen II (CII) is a well-known autoantigen in rheumatoid arthritis (RA). However, the direct effects of CII citrullination on cell behavior have not been described. To study whether citrullination of CII could affect cellular functions, we measured the adhesion of 3 different cell types (human Saos2 osteosarcoma cells, human synovial fibroblasts, and rat mesenchymal stem cells) with impedance-based technology. The binding of different collagen receptor integrins to citrullinated collagen was studied by CHO cell lines, each overexpressing 1 of the 4 human collagen receptors on the cell surface, and with solid-phase binding assays, using the recombinant human integrin α1I, α2I, α10I, and α11I domains. Collagen citrullination decreased the adhesion of synovial fibroblasts ∼50% (P<0.05) and mesenchymal stem cells ∼40% (P<0.05) by specifically decreasing the binding of integrins α10ß1 and α11ß1 to arginine-containing motifs, such as GFOGER. In contrast, citrullination had only a minor effect on the function of α1ß1 and α2ß1 integrins, which have been reported to play a critical role in regulating leukocyte function. Molecular modeling was used to explain the detected functional differences at the structural level. Given that the integrins regulate cell metabolism, proliferation, and migration, we suggest that collagen citrullination modifies the pathogenesis of RA. Here, CII citrullination was shown to decrease the survival of mesenchymal stem cells.

Integrins in periodontal disease.

Cell surface integrin receptors mediate cell adhesion, migration and cellular signaling in all nucleated cells. They are activated by binding to extracellular ligands or by intracellular proteins, such as kindlins that engage with their cytoplasmic tails. Cells in the periodontal tissues express several integrins with overlapping ligand-binding capabilities. A distinct phenotype in the periodontium has only been described for knockouts or mutations of three integrin subunits, α11, ß6 and ß2. Integrin α11ß1 appears to have some regulatory function in the periodontal ligament of continuously erupting incisors in mice. Integrin αvß6 is expressed in the junctional epithelium (JE) of the gingiva. Animals deficient in this receptor develop classical signs of periodontal disease, including inflammation, apical migration of the JE and bone loss, suggesting that it plays a role in the regulation of periodontal inflmmation, likely through activation of transforming growth factor-ß1. Lack of integrin activation in the JE is also associated with periodontitis. Patients with kindlin-1 mutations have severe early-onset periodontal disease. Finally, patients with mutations in the leukocyte-specific ß2 integrin subunit have severe periodontal problems due to lack of transiting neutrophils in the periodontal tissues.

Collagen XXII binds to collagen-binding integrins via the novel motifs GLQGER and GFKGER.

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 α2ß1 integrin. Solid-phase assays and surface plasmon resonance spectroscopy revealed a direct interaction between α2ß1 integrin and the motif GFKGER. In addition, immunohistochemical analysis demonstrated partial co-localization of collagen XXII, α2ß1 integrin and α11ß1 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.

Molecular mechanism of alpha2beta1 integrin interaction with human echovirus 1.

Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of alpha2beta1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the alpha2I domain. Furthermore, a mutation E336A in the alpha2 subunit, which inactivated alpha2beta1 as a collagen receptor, enhanced alpha2beta1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of alpha2beta1. This was supported by the fact that the integrin clustering by EV1 did not activate the p38 MAP kinase pathway, a signalling pathway that was shown to be dependent on E336-related conformational changes in alpha2beta1. Furthermore, the mutation E336A did neither prevent EV1 induced and alpha2beta1 mediated protein kinase C activation nor EV1 internalization. Thus, in its entry strategy EV1 seems to rely on the activation of signalling pathways that are dependent on alpha2beta1 clustering, but do not require the conformational regulation of the receptor.

Cellular signaling by collagen-binding integrins.

The four collagen-binding αI domain integrins form their own subgroup among cell adhesion receptors. The signaling functions of α1ß1 and α2ß1 integrins have been analyzed in many experimental models, whereas less studies are available about the more recently found α10ß1 and α11ß1 heterodimers. Interestingly, collagen binding by α1ß1 and α2ß1 often generates opposite cellular responses. For example α1ß1 has often been reported to promote cell proliferation and to suppress collagen synthesis, whereas α2ß1 can in many model systems inhibit growth and promote collagen synthesis. There are obviously cell type dependent factors modifying the signaling. Additionally the structure and the organization of collagenous matrix play a critic role. Many recent studies have also stressed the importance of the crosstalk between the integrins and other cell surface receptors.

Embigin deficiency leads to delayed embryonic lung development and high neonatal mortality in mice.

Embigin (Gp70), a receptor for fibronectin and an ancillary protein for monocarboxylate transporters, is known to regulate stem cell niches in sebaceous gland and bone marrow. Here, we show that embigin expression is at high level during early mouse embryogenesis and that embigin is essential for lung development. Markedly increased neonatal mortality of Emb-/- mice can be explained by the compromised lung maturation: in Emb-/- mice (E17.5) the number and the size of the small airways and distal airspace are significantly smaller, there are fewer ATI and ATII cells, and the alkaline phosphatase activity in amniotic fluid is lower. Emb-/- lungs show less peripheral branching already at E12.5, and embigin is highly expressed in lung primordium. Thus, embigin function is essential at early pseudoglandular stage or even earlier. Furthermore, our RNA-seq analysis and Ki67 staining results support the idea that the development of Emb-/- lungs is rather delayed than defected.

Collagen prolyl 4-hydroxylase isoenzymes I and II have sequence specificity towards different X-Pro-Gly triplets.

Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.

Novel α2ß1 integrin inhibitors reveal that integrin binding to collagen under shear stress conditions does not require receptor preactivation.

The interaction between α2ß1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm(2)). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent adhesion site motif in the α2I domain. The action of BTT-3033, unlike that of BTT-3034, was dependent on Tyr-285. In static conditions BTT-3034, but not BTT-3033, inhibited collagen binding by an α2 variant carrying a conformationally activating E318W mutation. Conversely, in under flow conditions (90 dynes/cm(2)) BTT-3033, but not BTT-3034, inhibited collagen binding by an α2 variant expressing E336A loss-of-function mutation. Thus, the binding sites for BTT-3033 and BTT-3034 are differentially available in distinct integrin conformations. Therefore, these sulfonamides can be used to study the biological role of different functional stages of α2ß1. Furthermore, only the inhibitor that recognized the non-activated conformation of α2ß1 integrin under shear stress conditions effectively blocked platelet adhesion, suggesting that the initial interaction between integrin and collagen takes place prior to receptor activation.

Intracellular degradation of multilabeled poly(ethylene imine)-mesoporous silica-silica nanoparticles: implications for drug release.

Mesoporous silica nanoparticles, MSNs, have emerged as an interesting carrier for drugs in vitro and in vivo. The particles are typically used in a surface functionalized form, where functional silanes or other covalently linked surface functions are used to provide anchoring sites for additional functionalities like targeting groups, imaging agents, and drugs. Here, we report results related to extra- and intracellular degradation of silica nanoparticles using multilabeled nonporous silica core-mesoporous silica shell-surface hyperbranched poly(ethylene imine) shell nanoparticles as model particles. Different fluorophores have been selectively covalently linked to different regions of the particles in order to study the particle degradation in detail under in vitro conditions in human SAOS-2 cells. A novel, quantitative method for nanoparticle degradation evaluation based on confocal fluorescence microscopy is applied. Our results suggest that the core-shell-shell MSNs degrade at a higher rate inside cells as compared to outside cells, which is of high importance for further application of this class of drug carriers.

Inhibition of TGF-ß signaling, invasion, and growth of cutaneous squamous cell carcinoma by PLX8394.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. The prognosis of patients with metastatic cSCC is poor emphasizing the need for new therapies. We have previously reported that the activation of Ras/MEK/ERK1/2 and transforming growth factor ß (TGF-ß)/Smad2 signaling in transformed keratinocytes and cSCC cells leads to increased accumulation of laminin-332 and accelerated invasion. Here, we show that the next-generation B-Raf inhibitor PLX8394 blocks TGF-ß signaling in ras-transformed metastatic epidermal keratinocytes (RT3 cells) harboring wild-type B-Raf and hyperactive Ras. PLX8394 decreased phosphorylation of TGF-ß receptor II and Smad2, as well as p38 activity, MMP-1 and MMP-13 synthesis, and laminin-332 accumulation. PLX8394 significantly inhibited the growth of human cSCC tumors and in vivo collagen degradation in xenograft model. In conclusion, our data indicate that PLX8394 inhibits several serine-threonine kinases in malignantly transformed human keratinocytes and cSCC cells and inhibits cSCC invasion and tumor growth in vitro and in vivo. We identify PLX8394 as a potential therapeutic compound for advanced human cSCC.