Nemosis, a novel way of fibroblast activation, in inflammation and cancer

Malignant cells when grown in suspension, as a rule, proliferate and can form spheroids that have been used as a model of tumor nodules, micrometastases and avascular tumors. In contrast, normal adherent cells cannot be stimulated to grow as multicellular aggregates. Now, recent results show that normal fibroblasts if forced to cluster (spheroid formation) do not grow but undergo a new pathway of cell activation (nemosis) leading to a massive proinflammatory, proteolytic and growth factor response. The clustering and activation are initiated by fibronectin-integrin interaction. The activated fibroblasts are able to modulate the behavior of cancer cells and, furthermore malignant cells boost this activation even further. In this model, the activation of fibroblasts terminates in programmed necrosis-like cell death. Activation of the tumor stroma, especially of fibroblasts, is of critical importance for tumor progression, although mechanisms leading to their activation are still largely uncharacterized. In summary, our results suggest that this kind of fibroblast activation (nemosis) may be involved in pathological conditions such as inflammation and cancer.     

Synergistic activity of fibronectin and fibroblast growth factor receptors on neuronal adhesion and neurite extension through extracellular signal-regulated kinase pathway

Fibroblast growth factor (FGF) is an important modulator of cell growth and differentiation of various cells including neuron. Cells need to adhere specifically to cellular and extracellular components of their environment to carry out diverse physiological functions. Here, we examined whether fibronectin (FN) and FGF can cooperate for neuronal adhesion and neurite outgrowth. Using recombinant FN peptide (FNIII9-10), we found that FNIII9-10-mediated adhesion promotes the effect of FGF1 on neurite outgrowth of PC12 cells, while FGF1 enhances the FNIII9-10-mediated neuronal adhesion of PC12 cells. This collaboration of FNIII9-10 and FGF1 was the result of the sustained activation of extracellular signal-regulated kinase (ERK)-type MAP kinase. Finally, the synergistic activity of FGF1 and FN was inhibited by PD98059, an MEK inhibitor. Taken together, these findings indicate that FN-mediated signaling can collaborate with FGFRs signaling for neurite outgrowth through selective activation of ERK-type MAP kinase in PC12 cells, and suggest that FN and FGF act in concert to regulate cell differentiation in the nervous system.     

MMP-2 functions as a negative regulator of chondrogenic cell condensation via down-regulation of the FAK-integrin beta1 interaction

Matrix metalloprotease-2 (MMP-2) has the capacity to degrade cartilage extracellular matrix molecules, the turnover of which is an essential event in chondrogenesis. Here, we investigated the functional role of MMP-2 in chondrogenesis of leg bud mesenchymal cells. Small interference RNA (siRNA)-mediated knockdown of mmp-2 promoted precartilage condensation and chondrogenesis. Treatment with bafilomycin A1, an MMP-2 activator, or GM6001, an MMP inhibitor, at the pre-condensation stage resulted in the inhibition or promotion of chondrogenesis, respectively. By comparison, treatment at the post-condensation stage had little or no effect on chondrogenesis. These results indicate that MMP-2 is involved in the regulation of cell condensation. Inhibition of MMP-2 activity by mmp-2 specific siRNA increased the protein level of fibronectin, and integrins alpha5 and beta1. The interaction between focal adhesion kinase (FAK) and integrin beta1 leading to tyrosine phosphorylation of FAK was also enhanced. Moreover, inactivation of p38MAPK down-regulated the level of MMP-2 mRNA and activity, and increased mesenchymal cell condensation in parallel with enhanced phosphorylation of FAK. Taken together, our data indicate that MMP-2 mediates the inhibitory signals of p38MAPK during mesenchymal cell condensation by functioning as a negative regulator of focal adhesion activity regulated by FAK via interactions with fibronectin through integrin beta1.     

Formation of mixed spheroids by a coculture of human cancerous and fibroblast cells

The coculture of fibroblasts with cancerous cells under the conditions which lead to spheroid formation, allowed the obtention of spheroids composed of a fibroblastic core surrounded by cancerous cells. The fibroblast core was labelled by hyaluronic acid and hyaluronectin. Hyaluronic acid concentration was also measured by enzymoimmunological assay in culture medium where it was found to accumulate during spheroid growth. The composite spheroid technique is a good model system for analysis of cancer cells-fibroblasts interaction in vitro.     

Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin

During early embryonic development, cranial neural crest cells emerge from the developing mid- and hindbrain. While numerous studies have focused on integrin involvement in trunk neural crest cell migration, comparatively little is known about mechanisms of cranial neural crest cell migration. We show that fibronectin, but not laminin, vitronectin, or type I collagen can support cranial neural crest cell migration and segmentation in vitro. These behaviors require both the RGD and "synergy" sites located within the central cell-binding domain of fibronectin. While these two sites are sufficient for cranial neural crest cell migration, we find that the second Heparin-binding domain of fibronectin can provide additional support for cranial neural crest cell migration in vitro. Finally, using a function blocking monoclonal antibody, we show that cranial neural crest cell migration on fibronectin requires the integrin alpha5beta1.     

The interaction between urokinase receptor and vitronectin in cell adhesion and signalling

The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells present in all tissue and organs. Cell-matrix interactions play fundamental roles during embryonic development, morphogenesis, tissue homoeostasis, wound healing, and tumourigenesis. Cell-matrix communication is kept in balance by physical contact and by transmembrane integrin receptors providing the dynamic link between the extracellular and intracellular environments through bi-directional signalling. The urokinase-type plasminogen activator receptor (uPAR) is a plasma membrane receptor overexpressed during inflammation and in almost all human cancers. One of its functions is to endorse ECM remodelling through the activation of plasminogen and downstream proteases, including matrix-metalloproteases (MMPs). Beside its role in ECM degradation, uPAR modulates cell-matrix contact through a direct engagement with the ECM component, vitronectin (Vn), and by regulating the activity state of integrins thus promoting or inhibiting integrin signalling and integrin-mediated cell adhesion to other ECM components, like fibronectin and collagen. In this review we have centred our attention on the non-proteolytic function of uPAR as a mediator of cell adhesion and downstream signalling.     

Multi-factorial modulation of IGD motogenic potential in MSF (Migration Stimulating Factor)

Migration Stimulating Factor (MSF) is a genetically truncated isoform of fibronectin (Fn). MSF is a potent stimulator of fibroblast migration, whereas full length Fn is devoid of motogenic activity. MSF and Fn contain four IGD motifs, located in the 3rd, 5th, 7th and 9th type I modules; these modules are referred to as ³FnI, ⁵FnI, ⁷FnI and ⁹FnI, respectively. We have previously reported that mutation of IGD motifs in modules ⁷FnI and ⁹FnI of MSF is sufficient to completely abolish the motogenic response of target adult skin fibroblasts. We now report that the IGD sequences in ³FnI and ⁵FnI are also capable of exhibiting motogenic activity when present within fragments of MSF. When present within ^1-5FnI, these sequences require the presence of serum or vitronectin for their motogenic activity to be manifest, whereas the IGD sequences in ⁷FnI and ⁹FnI are bioactive in the absence of serum factors. All MSF and IGD-containing peptides stimulated the phosphorylation of the integrin binding protein focal adhesion kinase (FAK) but did not necessarily affect migration. These results suggest that steric hindrance determines the motogenic activity of MSF and Fn, and that both molecules contain cryptic bioactive fragments.     

Collagen and fibronectin synthesis by trisomic and triploid fibroblasts from human spontaneous abortuses

Summary Collagen and fibronectin synthesis by trisomic and triploid fibroblasts derived from human spontaneous abortuses was studied. It was demonstrated that the level of fibronectin and collagen production in fibroblasts with trisomy 7, trisomy 9, and triploidy was reduced as compared with diploid cells. A correlation between this observation and an increased rate of intracellular ¹⁴C-procollagen degradation was also established for the anomalous strains. No difference in hydroxylation of ¹⁴C-proline residues in 1() and 2() collagen chains and no fluctuation in the collagen type (): type ratio was found in the strains with the abnormal karyotypes. It was concluded that differentiation of the abnormal fibroblasts was impaired. The data also favour the hypothesis that the deficiency of the fibroblasts in producing proteins may account for a variety of anatomic abnormalities of embryos.     

Density- and serum-dependent regulation of the Reck tumor suppressor in mouse embryo fibroblasts

Reck is a membrane-anchored glycoprotein identified as a transformation suppressor. Accumulating evidence indicates that Reck negatively regulates a wide spectrum of matrix metalloproteinases and is commonly down-regulated in a variety of malignant solid tumors. Physiological cues that regulate Reck expression, however, remained unknown. In this study, we found that Reck expression was up-regulated at high cell density, low serum, or after treatment with some kinase inhibitors, such as PP2 (Src inhibitor), LY294002 (PI3-kinase inhibitor), and PF573228 (FAK inhibitor), in mouse embryo fibroblasts. Curve fitting indicated that the levels of Reck protein and Reck mRNA are quadratic in the cell density. Other factors, including serum, extracellular matrix components (type I collagen and fibronectin), the kinase inhibitors, and some of their oncogenic targets (v-Src and PIK3CA mutants), modify the shape of the quadratic curve. Comparison of these modifications implicated Src in Reck down-regulation under sparse conditions, PI3-kinase in serum-induced Reck down-regulation, and FAK in Reck down-regulation at high cell density. Fibronectin and type I collagen down-regulated Reck, supporting the role of integrin-FAK signaling in Reck down-regulation at high cell density. Our study has revealed multiple signaling pathways impinging on Reck in cultured mouse embryo fibroblasts and sets a foundation for future studies to find effective Reck inducers of potential value in cancer therapy.     

Functional differences between integrin alpha4 and integrins alpha5/alphav in modulating the motility of human oral squamous carcinoma cells in response to the V region and heparin-binding domain of fibronectin

The high-affinity heparin-binding domain and the V region of fibronectin (FN) mediate invasion and migration of human oral squamous cell carcinoma (SCC) cells. We investigated the role of the alpha4, alpha5, and alphav integrin receptors--which are central to mediating interactions with these domains of FN--in regulating SCC cell migration. SCC cells expressed the alpha4, alpha5, and alphav integrin subunits on their surface, although alpha4 expression was low. Treatment with recombinant FN proteins containing an alternatively spliced V region (V+) and either an unmutated (H+) or a mutated, nonfunctional high-affinity heparin-binding domain (H-) increased expression of alpha5 and alphav and cell motility. Antisense alpha5 or alphav oligonucleotides inhibited cell motility stimulated by FN proteins, as did blocking antibodies to alpha5 or alphav. Blocking antibodies to alpha5 increased alphav and alpha4 levels, and blocking antibodies to alphav increased the levels of alpha5 and alpha4, without increasing cell motility. In contrast, an antisense alpha4 oligonucleotide and alpha4-blocking antibodies increased cell motility, especially migration stimulated by V+H+ and V+H- FN proteins. alpha4-Blocking antibodies alone increased motility, probably by inducing alpha5 and alphav expression. Transfection with alpha4 cDNA decreased cell motility and alpha5 and alphav expression. Thus, the increased motility induced by the FN protein is probably mediated by alphav and alpha5, whereas alpha4 downregulates this process in a transdominant fashion.     

Non-collagenous ECM proteins in blood vessel morphogenesis and cancer

Background

The extracellular matrix (ECM) is constituted by diverse composite structures, which determine the specific to each organ, histological architecture and provides cells with biological information, mechanical support and a scaffold for adhesion and migration. The pleiotropic effects of the ECM stem from the dynamic changes in its molecular composition and the ability to remodel in order to effectively regulate biological outcomes. Besides collagens, fibronectin and laminin are two major fiber-forming constituents of various ECM structures.

Scope of review

This review will focus on the properties and the biological functions of non-collagenous extracellular matrix especially on laminin and fibronectin that are currently emerging as important regulators of blood vessel formation and function in health and disease.

Major conclusions

The ECM is a fundamental component of the microenvironment of blood vessels, with activities extending beyond providing a vascular scaffold; extremely versatile it directly or indirectly modulates all essential cellular functions crucial for angiogenesis, including cell adhesion, migration, proliferation, differentiation and lumen formation. Specifically, fibronectin and laminins play decisive roles in blood vessel morphogenesis both during embryonic development and in pathological conditions, such as cancer.

General significance

Emerging evidence demonstrates the importance of ECM function during embryonic development, organ formation and tissue homeostasis. A wealth of data also illustrates the crucial role of the ECM in several human pathophysiological processes, including fibrosis, skeletal diseases, vascular pathologies and cancer. Notably, several ECM components have been identified as potential therapeutic targets for various diseases, including cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Transforming growth factor beta and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle.

Developmentally competent bovine blastocysts were produced by adding transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) to serum-free cultures of in vitro produced, 2-cell bovine embryos. The effects of TGF beta were evaluated because this growth factor signals synthesis and secretion of the extracellular matrix component fibronectin and its receptor. Previous investigations have demonstrated that fibronectin promotes early bovine embryo development in vitro. The effects of TGF beta can be potentiated by bFGF; bFGF itself is an effector of protein synthesis and a potent mitogen. A positive interaction between the 2 growth factors resulted in 38.8% of fertilized oocytes maturing beyond the 16-cell stage; of these, 24.6% formed blastocysts. Transfer of early blastocysts produced using serum-free medium supplemented with growth factors resulted in pregnancy in 3 of 9 recipients. These results support the hypothesis that TGF beta and bFGF act synergistically to promote development of bovine embryos beyond the "8-cell block" observed in vitro.     

Upregulation of fibronectin expression by COX-2 is mediated by interaction with ELMO1

Engulfment and cell motility 1 (ELMO1), a bipartite guanine nucleotide exchange factor (GEF) for the small GTPase Rac 1, was identified as a susceptibility gene for glomerular disease. Here, we reported that ELMO1 interacted with COX-2 in human mesangial cells. Furthermore, we identified ELMO1 as a posttranslational regulator of COX-2 activity. We demonstrated that COX-2 cyclooxygenase activity increased fibronectin promoter activity. The protein-protein interaction between ELMO1 and COX-2 increased the cyclooxygenase activity of COX-2 and, correspondingly, fibronectin expression. We also found that ET625, the dominant negative form of ELMO1 lacking Rac1 activity, interacted with COX-2, increased cyclooxygenase activity of COX-2 and enhanced COX-2-mediated fibronectin upregulation. To further rule out Rac1 as an ELMO1-mediated regulator of COX-2 activity, we employed the constitutive active Rac1, Rac1^Q63E, and demonstrated that Rac1 signaling has no effect on COX-2-mediated fibronectin promoter activity. These results suggest that ELMO1 contributes to the development of glomerular injury through serving as a regulator of COX-2 activity. The interaction of ELMO1 with COX-2 could play an important role in the development and progression of renal glomerular injury.     

Recombinant CBD-HepII polypeptide of fibronectin inhibits alphavbeta3 signaling and hematogenous metastasis of tumor

The interaction of integrin αvβ3 and its ligands are crucial for tumor metastasis. Recombinant CBD-HepII polypeptide of fibronectin, designated as CH50, suppressed the binding of tumor cells to ECM molecules, and abolished the promoting effect of soluble fibronectin and fibrinogen on tumor cell adhesion to ECM molecules. The underlying mechanisms involve the blockade and downregulation of αvβ3 and its co-receptor syndecan 1 by CH50. The activation of FAK, upregulation of cdc2, the production and activation of MMP-2 and MMP-9 by ECM molecules-stimulated tumor cells were inhibited by CH50. CH50 reduced the tumor cell arrest during blood flow, and also inhibited the invasive ability of tumor cells. The in vivo expressed CH50 suppressed the lung metastasis of circulating tumor cells, and prolonged the survival of mice after tumor cell inoculation. These findings suggest a prospective utility of CH50 in the gene therapy for prevention of tumor metastasis.     

Constitutively activated dystrophic muscle fibroblasts show a paradoxical response to TGF-β and CTGF/CCN2

Transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) have been described to induce the production of extracellular matrix (ECM) proteins and have been reported to be increased in different fibrotic disorders. Skeletal muscle fibrosis is a common feature of Duchenne muscular dystrophy (DMD). The mdx mouse diaphragm is a good model for DMD since it reproduces the muscle degenerative and fibrotic changes. Fibronectin (FN) and proteoglycans (PG) are some of the ECM proteins upregulated in dystrophic conditions. In view of understanding the fibrotic process involved in DMD we have isolated fibroblasts from dystrophic mdx diaphragms. Here we report that regardless of the absence of degenerative myofibers, adult mdx diaphragm fibroblasts show increased levels of FN and condroitin/dermatan sulfate PGs synthesis. Fibroblasts isolated from non fibrotic tissue, such as 1 week old mice diaphragms or skin, do not present elevated FN levels. Furthermore, mdx fibroblast conditioned media is able to stimulate FN synthesis in control fibroblasts. Autocrine TGF-β signaling was unaltered in mdx cells. When control fibroblasts are exposed to TGF-β and CTGF, FN increases as expected. Paradoxically, in mdx cells it decreases in a concentration dependent manner and this decrease is not due to a downregulation of FN synthesis. According to this data we hypothesize that a pathological environment is able to reprogram fibroblasts into an activated phenotype which can be maintained through generations.     

Human fibroblast culture on a crosslinked dermal porcine collagen matrix

The use of a novel porcine-derived collagen biomaterial as a dermal tissue engineering matrix was examined. The matrix is derived from porcine dermis, and is processed to retain the native collagen (Type 1) and elastin structure. Human primary fibroblasts were cultured on the matrix to examine its potential for creating a dermal replacement. Attachment of fibroblasts on the collagen was compared to tissue culture plastic and PET membranes. Cell proliferation was assessed using the MTT assay and DAPI staining. For seeding densities of 5×10⁴ and 1×10⁵ cells cm⁻², PET and plastic demonstrated >95% attachment of seeded numbers after 3 h. The collagen matrix reached levels >80% after 3–4 h with no influence of the seeding density. Matrix samples with perforating pores of 40 μm diameter were also studied. After 216 h culture in static culture, with media replacement every 3 days, the final cell numbers reached 2.1×10⁵ (perforated) and 2.0×10⁵ cells cm⁻² (unperforated). In comparison fibroblast culture in a perfusion bioreactor, with continuous media replacement, reached 2.3×10⁵ (unperforated) and 2.5×10⁵ cells cm⁻² (perforated) after 216 h.     

A Small Fibronectin-mimicking Protein from Bacteria Induces Cell Spreading and Focal Adhesion Formation

Fibronectin, a 250-kDa eukaryotic extracellular matrix protein containing an RGD motif plays crucial roles in cell-cell communication, development, tissue homeostasis, and disease development. The highly complex fibrillar fibronectin meshwork orchestrates the functions of other extracellular matrix proteins, promoting cell adhesion, migration, and intracellular signaling. Here, we demonstrate that CagL, a 26-kDa protein of the gastric pathogen and type I carcinogen Helicobacter pylori, mimics fibronectin in various cellular functions. Like fibronectin, CagL contains a RGD motif and is located on the surface of the bacterial type IV secretion pili as previously shown. CagL binds to the integrin receptor α₅β₁ and mediates the injection of virulence factors into host target cells. We show that purified CagL alone can directly trigger intracellular signaling pathways upon contact with mammalian cells and can complement the spreading defect of fibronectin^−/− knock-out cells in vitro. During interaction with various human and mouse cell lines, CagL mimics fibronectin in triggering cell spreading, focal adhesion formation, and activation of several tyrosine kinases in an RGD-dependent manner. Among the activated factors are the nonreceptor tyrosine kinases focal adhesion kinase and Src but also the epidermal growth factor receptor and epidermal growth factor receptor family member Her3/ErbB3. Interestingly, fibronectin activates a similar range of tyrosine kinases but not Her3/ErbB3. These findings suggest that the bacterial protein CagL not only exhibits functional mimicry with fibronectin but is also capable of activating fibronectin-independent signaling events. We thus postulate that CagL may contribute directly to H. pylori pathogenesis by promoting aberrant signaling cross-talk within host cells.     

Mediation of Rac1 activation by kindlin-2: an essential function in osteoblast adhesion, spreading, and proliferation

Kindlins are focal adhesion proteins that regulate integrin signaling. Although integrin activation is critical for bone development, little is known about the expression and role of kindlins in osteoblasts. We therefore investigated the function of kindlin-2 in osteoblast adhesion, spreading, and proliferation using small interfering RNA. In MC3T3-E1 cells, only kindlin-2 is highly expressed and localizes to focal adhesion. We found that kindlin-2 was involved in integrin activation in MC3T3-E1 cells and that kindlin-2 knockdown osteoblasts resulted in diminished cell adhesion, spreading, and proliferation. In this process, kindlin-2 knockdown impaired transient Rac1 activation, influencing Akt activation and AP-1 activity. In agreement with these data, pharmacological inhibition of Rac1 reduced MC3T3-E1 cell adhesion, spreading, and proliferation. Overall, these findings demonstrated that kindlin-2 governs Rac1 activation, which controls osteoblast function. Our findings provide the first insights concerning the function of kindlin-2 in osteoblast, and suggest that kindlin-2 is a critical mediator for osteoblast physiology.     

TCR-mediated Erk activation does not depend on Sos and Grb2 in peripheral human T cells

Sos proteins are ubiquitously expressed activators of Ras. Lymphoid cells also express RasGRP1, another Ras activator. Sos and RasGRP1 are thought to cooperatively control full Ras activation upon T-cell receptor triggering. Using RNA interference, we evaluated whether this mechanism operates in primary human T cells. We found that T-cell antigen receptor (TCR)-mediated Erk activation requires RasGRP1, but not Grb2/Sos. Conversely, Grb2/Sos—but not RasGRP1—are required for IL2-mediated Erk activation. Thus, RasGRP1 and Grb2/Sos are insulators of signals that lead to Ras activation induced by different stimuli, rather than cooperating downstream of the TCR.