Mouse Fibronectin Research Articles

Genomic Analysis of Vascular Invasion in HCC Reveals Molecular Drivers and Predictive Biomarkers.

Vascular invasion (VI) is a critical risk factor for HCC recurrence and poor survival. The molecular drivers of vascular invasion in HCC are open for investigation. Deciphering the molecular landscape of invasive HCC will help identify therapeutic targets and noninvasive biomarkers. To this end, we undertook this study to evaluate the genomic, transcriptomic, and proteomic profile of tumors with VI using the multiplatform cancer genome atlas (The Cancer Genome Atlas; TCGA) data (n=373). In the TCGA Liver Hepatocellular Carcinoma cohort, macrovascular invasion was present in 5% (n=17) of tumors and microvascular invasion in 25% (n=94) of tumors. Functional pathway analysis revealed that the MYC oncogene was a common upstream regulator of the mRNA, miRNA, and proteomic changes in VI. We performed comparative proteomic analyses of invasive human HCC and MYC-driven murine HCC and identified fibronectin to be a proteomic biomarker of invasive HCC (mouse fibronectin 1 [Fn1], P=1.7×10-11 ; human FN1, P=1.5×10-4 ) conserved across the two species. Mechanistically, we show that FN1 promotes the migratory and invasive phenotype of HCC cancer cells. We demonstrate tissue overexpression of fibronectin in human HCC using a large independent cohort of human HCC tissue microarray (n=153; P<0.001). Lastly, we showed that plasma fibronectin levels were significantly elevated in patients with HCC (n=35; mean=307.7 μg/mL; SEM = 35.9) when compared to cirrhosis (n=10; mean=41.8 μg/mL; SEM = 13.3; P<0.0001). Our study evaluates the molecular landscape of tumors with VI, identifying distinct transcriptional, epigenetic, and proteomic changes driven by the MYC oncogene. We show that MYC up-regulates fibronectin expression, which promotes HCC invasiveness. In addition, we identify fibronectin to be a promising noninvasive proteomic biomarker of VI in HCC.

Extracellular vesicles derived from injured vascular tissue promote the formation of tertiary lymphoid structures in vascular allografts.

Tertiary lymphoid structures (TLS) accumulate at sites of chronic injury where they function as an ectopic germinal center, fostering local autoimmune responses. Vascular injury leads to the release of endothelial‐derived apoptotic exosome‐like vesicles (ApoExo) that contribute to rejection in transplanted organs. The purpose of the study was to evaluate the impact of ApoExo on TLS formation in a model of vascular allograft rejection. Mice transplanted with an allogeneic aortic transplant were injected with ApoExo. The formation of TLS was significantly increased by ApoExo injection along with vascular remodeling and increased levels of antinuclear antibodies and anti‐perlecan/LG3 autoantibodies. ApoExo also enhanced allograft infiltration by γδT17 cells. Recipients deficient in γδT cells showed reduced TLS formation and lower autoantibodies levels following ApoExo injection. ApoExo are characterized by proteasome activity, which can be blocked by bortezomib. Bortezomib treated ApoExo reduced the recruitment of γδT17 cells to the allograft, lowered TLS formation, and reduced autoantibody production. This study identifies vascular injury‐derived extracellular vesicles (ApoExo), as initiators of TLS formation and demonstrates the pivotal role of γδT17 in coordinating TLS formation and autoantibody production. Finally, our results suggest proteasome inhibition with bortezomib as a potential option for controlling TLS formation in rejected allografts.

Improved differentiation of human enriched CD133+CD24+ renal progenitor cells derived from embryonic stem cell with embryonic mouse kidney-derived mesenchymal stem cells co-culture

End-stage renal disease (ESRD) is a major global public health issue. In the past decade, regenerative medicine and cell-based therapies were recommended for treatment of devastating diseases like ESRD. Renal progenitor (RP) cells are essential players in such treatment approaches. The major practical difficulties in application of RP cells are generation of these cells and preservation of their self-renewal capacity; also, they should lack identified appropriate cell surface markers. To identify and isolate RP cells, two cell surface markers namely, CD133 and CD24 were recently used. In this study, we used these markers to facilitate selection and purification of RP cells from embryoid bodies (EBs), and assessed the impact of the use of bFGF on frequency of CD133+CD24+ expression in cells presented in EBs. Moreover, following isolation of CD133+CD24+ cells from EBs, we evaluated the effect of embryonic, neonatal and adult mouse kidney-derived mesenchymal stem cells (E-KMSC, N-KMSC and A-KMSC respectively) and fibronectin on further differentiation of the sorted cells. Hence, we cultured undifferentiated human embryonic stem cells (hESCs) in suspension state in the presence or absence of bFGF and determined maximum number of CD133+CD24+ cells in bFGF-treated EBs on day 7. Then, we tested the effect of E-KMSC co-culture and seeding on fibronectin-coated plated on differentiation of the sorted cells into renal epithelial cells. Results revealed down-regulation of several RP cells, markers in CD133+CD24+ cells. In contrast, renal epithelial marker gene expressions were up-regulated after 7 days of co-culture with E-KMSC. Furthermore, fibronectin resulted in higher expression of renal epithelial markers compared to the E-KMSC co-cultured cells. All in all, bFGF could enhance the number of RP cells expressing CD133 and CD24 markers, in human EBs. We suggest E-KMSC and fibronectin as a promising supplementary factor to further induce differentiation of RP cells into renal epithelial cells.

Extracellular matrix mediators of metastatic cell colonization characterized using scaffold mimics of the pre-metastatic niche

Metastatic tumor cells colonize the pre-metastatic niche, which is a complex microenvironment consisting partially of extracellular matrix (ECM) proteins. We sought to identify and validate novel contributors to tumor cell colonization using ECM-coated poly(ε-caprolactone) (PCL) scaffolds as mimics of the pre-metastatic niche. Utilizing orthotopic breast cancer mouse models, fibronectin and collagen IV-coated scaffolds implanted in the subcutaneous space captured colonizing tumor cells, showing a greater than 2-fold increase in tumor cell accumulation at the implant site compared to uncoated scaffolds. As a strategy to identify additional ECM colonization contributors, decellularized matrix (DCM) from lungs and livers containing metastatic tumors were characterized. In vitro, metastatic cell adhesion was increased on DCM coatings from diseased organs relative to healthy DCM. Furthermore, in vivo implantations of diseased DCM-coated scaffolds had increased tumor cell colonization relative to healthy DCM coatings. Mass-spectrometry proteomics was performed on healthy and diseased DCM to identify candidates associated with colonization. Myeloperoxidase was identified as abundantly present in diseased organs and validated as a contributor to colonization using myeloperoxidase-coated scaffold implants. This work identified novel ECM proteins associated with colonization using decellularization and proteomics techniques and validated candidates using a scaffold to mimic the pre-metastatic niche. Statement of SignificanceThe pre-metastatic niche consists partially of ECM proteins that promote metastatic cell colonization to a target organ. We present a biomaterials-based approach to mimic this niche and identify ECM mediators of colonization. Using murine breast cancer models, we implanted microporous PCL scaffolds to recruit colonizing tumor cells in vivo. As a strategy to modulate colonization, we coated scaffolds with various ECM proteins, including decellularized lung and liver matrix from tumor-bearing mice. After characterizing the organ matrices using proteomics, myeloperoxidase was identified as an ECM protein contributing to colonization and validated using our scaffold. Our scaffold provides a platform to identify novel contributors to colonization and allows for the capture of colonizing tumor cells for a variety of downstream clinical applications.

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.

Plasma fibronectin concentration in inbred mouse strains

Plasma fibronectin concentration in inbred mouse strains

The Role of Akt-Forkhead Signaling in the Hematopoietic Stem-Progenitor Cell Differentiation: Expansion of Hematopoietic and Endothelial Progenitors from Akt-1 Null Marrows and Peripheral Blood Involving FKHRL1-Activation.

Akt is an important regulator of cell survival, growth and glucose metabolism in many cell types. In the previous annual meeting, we first reported the role of Akt in the hematopoietic stem cells with Akt-gene knockout mouse models. We described that Akt-signaling modulates the side population (SP) cell by directly regulating the molecular site of Bcrp1, one of the ATP-binding cassette transporters. Akt regulates many downstream targets through phosphorylation of a number of cellular substrates. FKHRL1, one of the FOXO subclass members of the forkhead box transcription factors, has a role in not only regulation of cell-cycle progression and cell survival phosphorylated but also control of cell-differentiation and transformation. FKHRL1 transcription-activities are inhibited by Akt, following induction of a prompt and sustained nuclear exclusion through phosphorylation. In this meeting, we will exhibit how Akt and its downstream signaling, FKHRL1 act during hematopoietic stem-progenitor cell differentiation with Akt1-null mouse studies and endothelial progenitor cell (EPC) assays. In bone marrow cells, a significant increase in formation of macrophage colony-forming units (CFU-M) and granulocyte-macrophage colony-forming units (CFU-GM) was seen in Akt1-null mice. Multiple growth factor responsive progenitor cultures were also more from Akt1-deficient marrows. Moreover, flow cytometry analysis showed the higher ratio of the lineage-negative progenitor cell-population in these marrows. Previously, we reported that bone marrow cellularity and the number of hematopoietic stem cells is normal in Akt1-null mice. These results indicate that lacking of Akt1-signaling leads to the proliferation potential of progenitor cells. Next, we analyzed the number of EPCs in Akt1-deficit mice from peripheral blood mononuclear cells (PBMCs) cultured on mouse fibronectin (FN)-coated dishes. DiI-Ac-LDL/lectin stained EPCs were detected after 10 days. Interestingly, Akt1-defecit mouse-EPCs were quite an increase in number, whereas few EPCs are usually detected from wild-type PBMCs. To address why EPCs were expanded, we used human EPC assay for analyzing signal transduction at detail. After attached on FN, circulating stem cells in PBMCs differentiated into EPCs with four different steps; foci-formation, sprout from the foci, migration for cord-like structure and maturation. Western blot analysis clearly showed that Akt was gradually activated during EPC-differentiation following the inactivation at the first step of differentiation. On the contrary, Akt-downstream targets, FKHRL1 and GSK3-β were inactivated through phosphorylation during differentiation. Immunofluorescent staining showed FKHRL1 was located in nucleus at the foci-formation and translocated into cytosol at the time of sprout from foci formation. Finally, lentivirus-mediated overexpression of Akt and FKHRL1 gene into mouse PBMCs showed FKHRL1-triple mutant, which is not phosphorylatable because of replacing three phosphorylation sites by alanine residues, significantly increased the number of EPCs, while constitutive active-Akt and dominant negative-FKHRL1 failed to. These data suggest that Akt-signaling has an important modulator of the hematopoietic stem-progenitor cell differentiation. FKHRL1 is involved in this mechanism.

Inhibition of human C4 step by mouse fibronectin

Inhibition of human C4 step by mouse fibronectin

Inhibition of human and mouse complement-dependent hemolytic activity by mouse fibronectin

Not only does mouse complement (C) have low hemolytic activity, but mouse serum has an inhibiting effect on hemolysis by human C. To purify and identify the putative mouse serum factor inhibiting human C activity, a sequential procedure of fractionated precipitation by PEG, followed by chromatographies with a heparin-Sepharose column, a phenyl-Sepharose column, a Protein G column, and a gel-filtration column was performed. The amino acid sequence analyses of two polypeptides obtained by digestion of the purified serum factor with TPCK-trypsin revealed that it was mouse fibronectin (FN). Highly purified mouse FN, but not human FN, has an inhibiting effect on human C-dependent hemolysis. Moreover, the hemolysis of sensitized rabbit erythrocytes by mouse C was also inhibited by the addition of mouse FN in a dose-dependent fashion, but not by the addition of human FN. These results suggest that FN is the putative internal C inhibitor in the mouse system.

Inhibition of complement-dependent hemolysis by mouse fibronectin

Inhibition of complement-dependent hemolysis by mouse fibronectin

Solution structure and dynamics of linked cell attachment modules of mouse fibronectin containing the RGD and synergy regions: comparison with the human fibronectin crystal structure

We report the three-dimensional solution structure of the mouse fibronectin cell attachment domain consisting of the linked ninth and tenth type III modules, mFnFn3(9,10). Because the tenth module contains the RGD cell attachment sequence while the ninth contains the synergy region, mFnFn3(9,10) has the cell attachment activity of intact fibronectin. Essentially complete signal assignments and approximately 1800 distance and angle restraints were derived from multidimensional heteronuclear NMR spectra. These restraints were used with a hybrid distance geometry/simulated annealing protocol to generate an ensemble of 20 NMR structures having no distance or angle violations greater than 0.3Åor 3°. Although the β-sheet core domains of the individual modules are well-ordered structures, having backbone atom rmsd values from the mean structure of 0.51(±0.12) and 0.40(±0.07)Å, respectively, the rmsd of the core atom coordinates increases to 3.63(±1.41)Åwhen the core domains of both modules are used to align the coordinates. The latter result is a consequence of the fact that the relative orientation of the two modules is not highly constrained by the NMR restraints. Hence, while structures of the β-sheet core domains of the NMR structures are very similar to the core domains of the crystal structure of hFnFn3(9,10), the ensemble of NMR structures suggests that the two modules form a less extended and more flexible structure than the fully extended rod-like crystal structure. The radius of gyration,Rg, of mFnFn3(9,10) derived from small-angle neutron scattering measurements, 20.5(±0.5)Å, agrees with the average Rgcalculated for the NMR structures, 20.4Å, and is ca 1Åless than the value of Rg calculated for the X-ray structure. The values of the rotational anisotropy,D∥/D⊥, derived from an analysis of 15N relaxation data, range from 1.7 to 2.1, and are significantly less than the anisotropy of 2.67 predicted by hydrodynamic modeling of the crystal coordinates. In contrast, hydrodynamic modeling of the NMR coordinates yields anisotropies in the range of 1.9 to 2.7 (average 2.4(±0.2)), with NMR structures bent by more than 20° relative the crystal structure having calculated anisotropies in best agreement with experiment. In addition, the relaxation parameters indicate that several loops in mFnFn3(9,10), including the RGD loop, are flexible on the nanosecond to picosecond time-scale. Taken together, our results suggest that, in solution, the limited set of interactions between the mFnFn3(9,10) modules position the RGD and synergy regions to interact specifically with cell surface integrins, and at the same time permit sufficient flexibility that allows mFnFn3(9,10) to adjust for some variation in integrin structure or environment.

Human papillomavirus type 16 E6 protein transcriptionally modulates fibronectin gene expression by induction of protein complexes binding to the cyclic AMP response element

Although human papillomavirus type 16 (HPV16) E6 protein has a transcription-modulatory activity for a wide variety of viral promoters, a cellular target for this activity of E6 has not yet been identified. In this study, using differential hybridization, we identified a mouse fibronectin (FN) gene as a putative cellular target whose expression is up-regulated by E6. Chloramphenicol acetyltransferase (CAT) assays with mouse and rat FN promoter-CAT fusion constructs indicated that HPV16 E6 transactivates the FN promoters in a p53-independent manner. Deletion and site-specific mutation analyses revealed that transactivation by HPV16 E6 depends upon a cyclic AMP response element (CRE) located at -160 relative to the start site of transcription. Gel retardation assays demonstrated that nuclear extracts from the HPV16 E6-expressing cells, compared to those from parental 10T1/2 cells, have increased binding activity to the CRE. Antibodies against c-Jun and ATF-2 disrupted this binding activity. These data indicate that HPV16 E6 transcriptionally modulates FN gene expression via the CRE by inducing the binding of the protein complexes, probably including c-Jun and ATF-2, to the CRE.

Expression of the mouse fibronectin gene and fibronectin-lacZ transgenes during somitogenesis.

Fibronectins (FNs) are essential for the proper development of embryonic mesenchymal tissues. A lacZ reporter gene has been fused to 4.9 kbp of DNA from the rat FN gene 5' flanking region, and this construct has been microinjected into fertilized mouse embryos to investigate the cis elements needed for the temporal and spatial regulation of FN in vivo. Histochemical staining of embryos for beta-galactosidase activity demonstrated that four independent lines shared a specific pattern of lacZ expression, reflecting the activity of the fibronectin sequences contained within the transgene. Specifically, somites stained positively for lacZ, but expression was spatially and temporally non-uniform, with higher levels in more caudal somites after a total of ca. 13 somite pairs had formed. This rostral-caudal gradient of lacZ expression in somites of embryos beyond this stage resembled the distribution of endogenous FN mRNA, as detected by whole mount in situ hybridization. The transgene was not expressed in the developing heart where endogenous FN mRNA was detected. Unexpectedly, highly localized staining was observed within the neural tube beginning at ca. E10-10.5, and two of the lines exhibited additional areas of staining due to the individual integration sites. Thus, the 4.9 kbp FN fragment appears to recapitulate closely the complex pattern of FN expression observed during somitogenesis. A smaller fragment of 0.9 kbp also directed lacZ expression in caudal somites at E9.5, suggesting that these sequences are sufficient to establish the spatio-temporal pattern.

Survival of tyrosine hydroxylase-positive cells and behavioral recovery after introduction of TH gene using adenoviral vector

We have evaluated neurite outgrowth from mesencephalic tyrosine hydroxylase-positive neurons grown in vitro on different substrates. Cultures of ventral mesencephalon from rat embryos (E13) were plated on plastic dishes coated with the following substrates: L1, L2/HNK-1 “residual” (mainly J1/160 but also tenascin), MAG antigens from mouse brains, laminin, fibronectin, poly-l-lysine, RGD peptide, and plastic alone. After 3, 4, and 6 days in vitro, the cultures were stained using an antibody against tyrosine hydroxylase (TH), and the length of TH-positive neurites was measured by computer-assisted image analysis in a double-blind fashion. L1 antigen had a significant positive effect on neurite outgrowth compared to the other substrates studied. Laminin and fibronectin were also favorable substrates. In cultures treated with cytosine arabinoside to prevent mitoses and glial proliferation, the positive effect of L1 was abolished, but laminin still had a stimulatory effect. These data indicate that L1 may be indirectly involved in differentiation or axonal elongation of substantia nigra dopaminergic neurons and suggest a complex effect involving both neurons and glia on dopaminergic neurite development.

CLONING OF THE MOUSE FIBRONECTIN V‐REGION AND VARIATION OF ITS SPLICING PATTERN IN EXPERIMENTAL IMMUNE COMPLEX GLOMERULONEPHRITIS

Increased mRNA and protein expression of extracellular matrix (ECM) components, including fibronectin, occurs during the development of glomerulonephritis and glomerulosclerosis in immunologically mediated kidney diseases. However, in addition to these quantitative changes in ECM expression, qualitative changes in these molecules may contribute to malformations in the composition of the glomerular matrix. These qualitative changes may include alterations in the splicing pattern of the V-region of fibronectin, since this region plays a role in its accumulation. The splicing patterns of this region have been studied in chronic graft-versus-host disease (GvHD) in mice, a model of lupus nephritis, and in chronic serum sickness (CSS) in rats, a model of immune complex nephritis. Cloning of the mouse fibronectin V-region from kidney tissue revealed 96·1 per cent homology with the corresponding domain in rat fibronectin. PCR (polymerase chain reaction) analysis of RNA from isolated glomeruli revealed three isoforms of this region in both mouse and rat fibronectin, namely inclusion or exclusion of the whole region, or exclusion of only the CS1 domain. In both models, increased exclusion of the V-region was observed early in the disease. However, in GvHD the splicing pattern returned to normal, whereas in CSS the shift persisted during the course of the experiment. Differentiated expression of fibronectin isoforms may exert an important effect on the structure and biological function of the glomerulus and may thus play a role in the development of glomerulonephritis and glomerulosclerosis.

CLONING OF THE MOUSE FIBRONECTIN V-REGION AND VARIATION OF ITS SPLICING PATTERN IN EXPERIMENTAL IMMUNE COMPLEX GLOMERULONEPHRITIS

Increased mRNA and protein expression of extracellular matrix (ECM) components, including fibronectin, occurs during the development of glomerulonephritis and glomerulosclerosis in immunologically mediated kidney diseases. However, in addition to these quantitative changes in ECM expression, qualitative changes in these molecules may contribute to malformations in the composition of the glomerular matrix. These qualitative changes may include alterations in the splicing pattern of the V-region of fibronectin, since this region plays a role in its accumulation. The splicing patterns of this region have been studied in chronic graft-versus-host disease (GvHD) in mice, a model of lupus nephritis, and in chronic serum sickness (CSS) in rats, a model of immune complex nephritis. Cloning of the mouse fibronectin V-region from kidney tissue revealed 96.1 per cent homology with the corresponding domain in rat fibronectin. PCR (polymerase chain reaction) analysis of RNA from isolated glomeruli revealed three isoforms of this region in both mouse and rat fibronectin, namely inclusion or exclusion of the whole region, or exclusion of only the CS1 domain. In both models, increased exclusion of the V-region was observed early in the disease. However, in GvHD the splicing pattern returned to normal, whereas in CSS the shift persisted during the course of the experiment. Differentiated expression of fibronectin isoforms may exert an important effect on the structure and biological function of the glomerulus and may thus play a role in the development of glomerulonephritis and glomerulosclerosis.

NEGATIVE REGULATION OF FIBRONECTIN GENE-TRANSCRIPTION BY EJ-HA-RAS AND C-SIS ONCOGENES IN BALB/C 3T3 CELLS

Down-regulation of fibronectin gene expression at cell surfaces by various oncogenes is an important phenotypic change of many transformed cells. Mechanisms effecting this loss of cell surface fibronectin remain poorly understood. An isogenic mouse fibroblast system, Balb/c 3T3, was used to investigate the effect of oncogene transformation on fibronectin gene transcription. Fibronectin mRNA levels were shown to decrease by >80% when 3T3 cells became stably transformed by EJ-Ha-ras or c-sis oncogenes. The mouse fibronectin gene promoter was subsequently cloned from a mouse genomic library and inserted into reporter gene constructs. Transient transfection assays with the 1.2 kb fibronectin promoter demonstrated that its activity was also decreased in the two transformed cell lines to a similar extent as fibronectin mRNA levels. Co-transfections into 3T3 cells with both the promoter construct and either Ha-ras or c-sis expression constructs provided further support for negative regulation of fibronectin gene transcription by oncogene expression. Potential mechanisms for such transcriptional regulation are proposed based on deletion analysis of the fibronectin promoter. These data suggest that down-regulation of fibronectin gene transcription can contribute directly to the loss of cell surface fibronectin in transformed murine fibroblasts.

Fibronectin gene expression in proliferating, quiescent, and SV40-infected mouse kidney cells

To study alterations in cellular gene expression in mouse kidney cell cultures infected with simian virus 40 (SV40) or polyomavirus, we performed a differential screening of a mouse kidney cDNA library with probes prepared from mRNAs of virus-infected and mock-infected cells. We isolated and characterized cDNA recombinant pKT13 which detected increased mRNA levels in infected cells. Sequence analysis of pKT13 revealed close to 100% homology with the 3′-end of mouse fibronectin (FN) mRNA. Since primary cultures of baby mouse kidney cells have been extensively characterized in our laboratories, we studied FN gene expression at different stages of uninfected and virus-infected cultures. High levels of FN and of its mRNA were found in the kidneys of suckling mice, while in primary cultures of proliferating epithelial kidney cells the expression of FN was very low until the cultures became confluent. Thereafter FN increased and reached high levels in cells which were irreversibly arrested in phase G 0 and which had apparently exhausted their finite division potential. Infection of confluent cultures with polyomavirus or SV40 resulted in a further stimulation of FN gene expression. However, during abortive infection with SV40, FN mRNA and FN levels decreased with emergence of transformed cells and were low in an established SV40-transformed mouse kidney cell line. These changes in FN gene expression suggest that high levels of FN might be indicative in vivo for terminal differentiation and in vitro for cellular senescence.

Nephrotoxic antiserum identifies a beta 1-integrin on rat glomerular epithelial cells

A postulated mechanism of immune glomerular injury is a direct interaction between antibody and glomerular epithelial cell (GEC) surface antigens. To explore this hypothesis, we examined the interaction of the noncomplement-fixing gamma 2-subclass of sheep anti-rat nephrotoxic serum (NTS), which causes immediate complement- and neutrophil-independent proteinuria in vivo, with rat GECs in culture. Reactivity of NTS with GEC surface antigens was determined by positive immunofluorescence of GEC plasma membranes and by the ability of NTS-coated tissue culture wells to provide an adhesive substrate for GECs. NTS immunoprecipitated two proteins (135 and 118 kDa) from surface-labeled GECs. Proteins of similar molecular mass were precipitated by a polyclonal rabbit antibody that identifies the beta 1-integrin chain of the mouse fibronectin receptor (anti-FnR). In addition, NTS identified similarly sized bands on Western blot analysis of cell membranes from isolated rat glomeruli. Similar reactivity was eluted from the glomeruli of proteinuric rats injected with NTS. NTS significantly inhibited GEC adhesion to laminin, types I and IV collagen, and fibronectin and prevented GEC spreading on types I and IV collagen. Anti-FnR similarly inhibited GEC adhesion. Cell viability was not affected. These results show that NTS recognizes a pair of GEC surface proteins that have the characteristics of an alpha- and beta 1-integrin and, at low concentrations, disrupt cell-matrix interactions.

Recombinant carboxyl-terminal fibrin-binding domain of human fibronectin expressed in mouse L cells.

The carboxyl-terminal fibrin-binding domain, Fib2, of human fibronectin was expressed in mouse L cells as a fusion protein with the signal sequence of human protein C inhibitor. The recombinant Fib2 (rFib2) protein synthesized by transfected cells retained the ability to form dimers with each other or with mouse fibronectin subunits and was secreted to the medium after extensive glycosylation. Only a small fraction of the secreted protein was incorporated into the pericellular matrix. Interestingly, the secreted rFib2 protein displayed a remarkable heterogeneity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, giving rise to a broad band corresponding to Mr of 60,000-90,000. The heterogeneity was eliminated mostly by treatment with neuraminidase and further by treatment with endo-alpha-N-acetylgalactosaminidase. Treatment with peptide:N-glycosidase F did not alter the heterogeneity of the protein, indicating that differential sialylation of O-linked, but not N-linked, glycans is largely responsible for the apparent heterogeneity. The presence of O-linked but absence of N-linked glycans was further supported by the observations that peanut agglutinin specifically bound to the desialylated rFib2 protein, whereas neither concanavalin A nor lentil lectin bound to the protein irrespective of prior neuraminidase treatment. Since the apparent heterogeneity of the rFib2 protein was only observable with the secreted, but not the cytoplasmic form, sialylation of O-linked glycans may be essential for, or regulate as a rate-limiting step, the transit of the recombinant protein to the extracellular space.