Alternative Splicing Of Fibronectin Research Articles

Formation of an invasion-permissive matrix requires TGFβ/SNAIL1-regulated alternative splicing of fibronectin

BackgroundAs in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFβ, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFβ target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses.MethodsWe used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis.ResultsIn silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFβ-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix that does not sustain TGFβ-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis.ConclusionsOur results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.

Role of pericytes in the development of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is caused by loss-of-function mutations in CCM1, CCM2, or CCM3 genes of endothelial cells. It is characterized by pericyte deficiency. However, the role of pericytes in CCMs is not yet clarified. We found pericytes in Cdh5Cre ERT2 ;Ccm1 fl/fl (Ccm1 ECKO ) mice had a high expression of PDGFRβ. The inhibition of pericyte function by CP-673451 aggravated the CCM lesion development. RNA-sequencing analysis revealed the molecular traits of pericytes, such as highly expressed ECM-related genes, especially Fn1. Furthermore, KLF4 coupled with phosphorylated SMAD3 (pSMAD3) promoted the transcription of fibronectin in the pericytes of CCM lesions. RGDS peptide, an inhibitor of fibronectin, decreased the lesion area in the cerebella and retinas of Ccm1 ECKO mice. Also, human CCM lesions had abundant fibronectin deposition, and pSMAD3- and KLF4-positive pericytes. These findings indicate that pericytes are essential for CCM lesion development, and fibronectin intervention may provide a novel target for therapeutic intervention in such patients.

Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow.

Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.

Nucleolin Interacts and Co-Localizes with Components of Pre-Catalytic Spliceosome Complexes

Nucleolin is an RNA binding protein that is involved in many post-transcriptional regulation steps of messenger RNAs in addition to its nucleolar role in ribosomal RNA transcription and assembly in pre-ribosomes. Acetylated nucleolin was found to be associated with nuclear speckles and to co-localize with the splicing factor SC35. Previous nuclear pull down of nucleolin identified several splicing components and factors involved in RNA polymerase II transcription associated with nucleolin. In this report, we show that these splicing components are specifics of the pre-catalytic A and B spliceosomes, while proteins recruited in the Bact, C and P complexes are absent from the nucleolin interacting proteins. Furthermore, we show that acetylated nucleolin co-localized with P-SF3B1, a marker of co-transcriptional active spliceosomes. P-SF3B1 complexes can be pulled down with nucleolin specific antibodies. Interestingly, the alternative splicing of Fibronectin at the IIICS and EDB sites was affected by nucleolin depletion. These data are consistent with a model where nucleolin could be a factor bridging RNA polymerase II transcription and assembly of pre-catalytic spliceosome similarly to its function in the co-transcriptional maturation of pre-rRNA.

Histone Deacetylase Activity Modulates Alternative Splicing

There is increasing evidence to suggest that splicing decisions are largely made when the nascent RNA is still associated with chromatin. Here we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection. Using splicing-sensitive microarrays, we identified ∼700 genes whose splicing was altered after HDAC inhibition. We provided evidence that HDAC inhibition induced histone H4 acetylation and increased RNA Polymerase II (Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduced co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. We further showed that the depletion of HDAC1 had similar effect on fibronectin alternative splicing as global HDAC inhibition. Importantly, this effect was reversed upon expression of mouse HDAC1 but not a catalytically inactive mutant. These results provide a molecular insight into a complex modulation of splicing by HDACs and chromatin modifications.

Identification of a pre-mRNA splicing factor, arginine/serine-rich 3 (Sfrs3), and its co-expression with fibronectin in fetal and postnatal rabbit airway mucosal and skin wounds

Fibronectin (FN) is a multi-functional, adhesion protein and involved in multi-steps of the wound healing process. Strong evidence suggests that FN protein diversity is controlled by alternative RNA splicing; a coordinated transcription and RNA processing that is development-, age-, and tissue/cell type-regulated. We previously demonstrated that fetal rabbit airway mucosal healing is regenerative and scarless. Expression, regulation, and biological function of the FN gene and various spliced forms in this model are unknown. Airway and skin incisional wounds were made in fetal (gestation days 21-23), weanling (4-6 weeks) and adult (>6 months) rabbits. Non-wounded and wounded tissues were collected at 12 h (all age groups), 24 h and 48 h (weanling only) post-wounding. Expression profiles were obtained using mRNA differential display and cDNAs of interest were cloned, sequenced and validated by real-time PCR. Here, we report two rabbit cDNAs that showed similar expression patterns after wounding. One encodes a rabbit fibronectin gene, Fn1, and another shares a high sequence homology to a human pre-mRNA splicing factor, arginine/serine-rich 3 (Sfrs3), coding for a RNA binding protein, SRp20. Both Fn1 and Sfrs3 mRNAs were suppressed in fetal wounds but induced in postnatal wounds 12 h post-wounding. The increased levels of both Fn1 and Sfrs3 transcripts were sustained up to 48 h in weanling airway mucosal wounds. The augmentations of the two genes in postnatal airway mucosal wounds were more prominent than that in skin wounds, indicating that the involvement of Sfrs3 and Fn1 genes in postnatal airway mucosal wounds is tissue-specific. Literature provides evidence that SRp20 is indeed involved in the alternative splicing of FN and that the embryonic FN variants reappear during adult wound healing. A connection between the enhanced molecular activity of Sfrs3 and the regulation of the FN gene expression through alternative splicing during the early events of postnatal airway mucosal wound repair was proposed.

Mammary Epithelial-Mesenchymal Interaction Regulates Fibronectin Alternative Splicing via Phosphatidylinositol 3-Kinase

The way alternative splicing is regulated within tissues is not understood. A relevant model of this process is provided by fibronectin, an important extracellular matrix protein that plays a key role in cell adhesion and migration and contains three alternatively spliced regions known as EDI, EDII, and IIICS. We used a cell culture system to simulate mammary epithelial-stromal communication, a process that is crucial for patterning and function of the mammary gland, and studied the effects of extracellular signals on the regulation of fibronectin pre-mRNA alternative splicing. We found that soluble factors from a mammary mesenchymal cell-conditioned medium, as well as the growth factors HGF/SF (hepatocyte growth factor/scatter factor), KGF (keratinocyte growth factor), and aFGF (acidic fibroblast growth factor), stimulate EDI and IIICS but not EDII inclusion into fibronectin mRNA in the mammary epithelial cell line SCp2, favoring fibronectin isoforms associated with proliferation, migration, and tissue remodeling. We explored the signaling pathways involved in this regulation and found that the mammary mesenchymal cell-conditioned medium and HGF/SF act through a phosphatidylinositol 3-kinase-dependent cascade to alter fibronectin alternative splicing. This splicing regulation is independent from promoter structure and de novo protein synthesis but does require two exonic elements within EDI. These results shed light on how extracellular stimuli are converted into changes in splicing patterns.

Alternative splicing of fibronectin: a mouse model demonstrates the identity of in vitro and in vivo systems and the processing autonomy of regulated exons in adult mice.

We have designed a novel approach using genetically engineered mice to make a systematic study of the EDA exon regulation of the fibronectin gene during development and aging. The genome of the mice was modified either by optimization of the EDA natural splice sites or by deleting the EDA region. The previous in vitro observation that the optimization of the splicing sites leads to constitutive inclusion of the EDA exon was confirmed in our animal model. In fact, all the adult tissues of the genetically modified mice showed only EDA(+) FN mRNA, demonstrating the fidelity of in vitro models, despite of the development- and aging-regulated splicing regulation of the EDA exon, and regardless of the presence of exonic elements described within the exon. This result indicates that the splicing regulatory elements of the EDA exon are dispensable in the presence of consensus splicing sites. Moreover, we demonstrate the autonomy of both the EDB and the IIICS alternatively spliced regions in adult mice lacking regulation of the alternative splicing at the EDA exon. We also show here the tight splicing regulation of all three alternative spliced regions of the FN gene at different time-points during development and aging of mice.

Regulation of fibronectin alternative splicing by a basement membrane-like extracellular matrix

Hepatocytes are the source of plasma fibronectin (FN) which lacks the alternatively spliced EDI segment, distinctive of oncofetal FN. When hepatic or other epithelial cells are cultured on plastic, EDI inclusion is triggered. Here we report that EDI inclusion is inhibited when hepatic cells are cultured on a basement membrane-like extracellular matrix (ECM), demonstrating a new role for the ECM in the control of gene expression. The effect is duplicated by collagen IV and laminin but not by collagen I; is not observed with another alternatively spliced FN exon (EDII); and correlates with a decrease in cell proliferation, consistently with high EDI inclusion levels observed in many physiological and pathological proliferative processes.

α4 Integrin Is Expressed during Peripheral Nerve Regeneration and Enhances Neurite Outgrowth

We have shown previously that repair in the peripheral nervous system is associated with a reversion to an embryonic pattern of alternative splicing of the extracellular matrix molecule fibronectin. One of the consequent changes is a relative increase in the number of fibronectins expressing the binding site for alpha4 integrins. Here we show that alpha4 integrins are expressed on dorsal root ganglion neuron cell bodies and growth cones in the sciatic nerve during regeneration and that the interaction of alpha4 integrin with alternatively spliced isoforms of recombinant fibronectins containing the alpha4 binding site enhances neurite outgrowth in dorsal root ganglion neurons. The pheochromocytoma (PC12) neuronal cell line, which normally extends neurites poorly on fibronectin, does so efficiently when alpha4 is expressed in the cells. Experiments using chimeric integrins expressed in PC12 cells show that the alpha4 cytoplasmic domain is necessary and sufficient for this enhanced neurite outgrowth. In both dorsal root ganglion neurons and PC12 cells the alpha4 cytoplasmic domain is tightly linked to the intracellular adapter protein paxillin. These experiments suggest an important role for alpha4 integrin and paxillin in peripheral nerve regeneration and show how alternative splicing of fibronectin may provide a mechanism to enhance repair after injury.

Evidence for a role of the alternatively spliced ED-I sequence of fibronectin during ovarian follicular development.

This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovarian follicle. In particular, we were interested in determining the effect of the ED-I (also termed ED-A) type III repeat, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I+ FN, whereas total FN levels remained relatively constant. ED-I+ FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological role for ED-I+ FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 10-fold decrease in the relative proportion of the ED-I region. In contrast, transforming growth factor-beta elicited a 2-fold stimulation of overall FN synthesis and a 4-fold increase in the synthesis of ED-I containing FN. This effect was evident at the protein (Western blots) and messenger RNA (Northern blots) levels. Although a negative correlation (P < 0.001) was detected between ED-I+ FN and estradiol levels in follicular fluid, this steroid was unable to modulate in vitro the alternative splicing of FN. A possible mitogenic effect of ED-I+ FN was suggested by the observation that a recombinant peptide corresponding to the ED-I domain stimulated DNA synthesis in a bovine granulosa cell line (BGC-1), whereas a peptide corresponding to the flanking type III sequences had no effect. The hypothesis of ED-I+ FN as a growth regulatory factor was further strengthened by the fact that depletion of FN from BGC-1-conditioned medium, which contained ED-I+ FN, abrogated its mitogenic activity, whereas plasma FN was without effect. We propose that changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.

Regulation of fibronectin alternative splicing during peripheral nerve repair

Regulation of fibronectin alternative splicing during peripheral nerve repair

Regulation of alternative pre-mRNA splicing by a novel repeated hexanucleotide element.

The alternatively spliced exon EIIIB is regulated in a cell type-specific manner in the rat fibronectin gene. Splicing of EIIIB into fibronectin mRNA is dependent on sequences in the intron immediately downstream of EIIIB. We show that a short, highly repeated TGCATG motif in this intron is important for cell type-specific recognition of EIIIB as an exon. This motif enhances usage of the EIIIB 5' splice site; furthermore, this repeated TGCATG sequence can activate an alternatively spliced exon in the unrelated rat preprotachykinin pre-mRNA. Interestingly, this sequence can also be found within cis-acting elements identified previously in other alternatively spliced genes. This short repeated TGCATG motif is therefore a cell type-specific element that, in addition to controlling fibronectin alternative splicing, may participate in the regulation of other alternative RNA processing events.

Alternative splicing of fibronectin pre-mRNA during aging.

Alternative splicing of fibronectin pre-mRNA during aging.

α5 Integrin subunit expression changes during myogenesis

Fibronectin and its cellular receptor, the α 5 β 1 integrin, are involved in the transmembrane signalling events that control muscle cell differentiation. In this study, the expression of the α 5 integrin subunit was followed by reverse transcription-polymerase chain reaction (RT-PCR) to determine alterations during myogenesis. In studies of murine muscle, we found a 90% reduction in the level of the α 5 integrin subunit mRNA during early postnatal development. Concurrently, the fibronectin alternative splicing pattern changed markedly in the EIIIB and V exons. In-vitro analyses of these molecules during myoblast differentiation revealed changes that followed trends similar to those observed in vivo, although of lesser magnitude. These observations imply an important role of fibronectin and the α 5 integrin subunit in muscle development.

Alternative splicing of fibronectin: Three variants, three functions

Fibronectin (FN) is a multi-functional extracellular matrix protein required for cell adhesion and migration, blood clotting, wound healing, and oncogenic transformation. The functional complexity is paralleled by structural diversity in that multiple forms of FN are generated by cell type-specific alternative splicing. In the rat, up to 12 different combinations of the three alternatively spliced segments (EIIIA, EIIIB, and the V region) are produced. What effects do these segments have on FN function? Recently, progress has been made in the identification of specific activities for the three variants of the V region, V120, V95, and V0. FN-mediated cell adhesion, FN synthesis and secretion, and incorporation into blood clots are differentially affected by these isoforms. These results suggest that cellular behavior is modulated by environmental cues provided by different types and proportions of alternatively spliced FN variants.

Polarized regulation of fibronectin secretion and alternative splicing by transforming growth factor

Fibronectin is a multifunctional protein that is synthesized in several different forms that result from alternative splicing of mRNA. Although expression of splicing variants appears to be both developmentally regulated and tissue-specific, the functional significance of these isoforms is largely unknown. We found that cultured airway epithelial cells vectorially secrete two distinct species of fibronectin, one which contains the alternatively spliced EIIIA region (EIIIA+) and one in which the EIIIA segment is spliced out (EIIIA-). Fibronectin containing the EIIIA region is preferentially secreted apically. Although both apical and basal stimulation with transforming growth factor beta 1 increased fibronectin synthesis, the secretory response differed depending on which surface was being stimulated. Apical secretion of fibronectin and expression of EIIIA+ fibronectin mRNA increased only after apical stimulation. These data demonstrate a novel mechanism for the polarized regulation of targeted secretion and alternative splicing of fibronectin and suggest that the EIIIA segment may act as a targeting signal for the vectorial secretion of fibronectin.