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• W2134839779 abstract "The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA+FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA+FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues. The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA+FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA+FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues. Fibronectins (FN) 3The abbreviations used are: FNfibronectinpFNplasma FNEDAextra domain AIIICStype III homologies connecting segmentWTwild typeRT-PCRreverse transcription PCR. are a family of multifunctional glycoproteins known to play key roles in fundamental processes related to adhesive and migratory behavior of cells, such as embryogenesis, malignancy, homeostasis, wound healing, and maintenance of tissue integrity (1Hynes R.O. Fibronectins. Springer-Verlag, New York1990Crossref Google Scholar). FN generates protein diversity as a consequence of alternative processing of a single primary transcript at three different sites, the extra domain A (EDA), the extra domain B (EDB), and the type III homologies connecting segment (IIICS) (2Kornblihtt A.R. Vibe-Pedersen K. Baralle F.E. EMBO J. 1984; 3: 221-226Crossref PubMed Scopus (143) Google Scholar, 3Gutman A. Kornblihtt A.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7179-7182Crossref PubMed Scopus (100) Google Scholar, 4Schwarzbauer J.E. Tamkun J.W. Lemischka I.R. Hynes R.O. Cell. 1983; 35: 421-431Abstract Full Text PDF PubMed Scopus (483) Google Scholar). Two major forms of FN exist, plasma FN (pFN) and cellular FN. pFN is a soluble dimeric form that is secreted into the bloodstream by hepatocytes (5Owens M.R. Cimino C.D. Blood. 1982; 59: 1305-1309Crossref PubMed Google Scholar, 6Tamkun J.W. Hynes R.O. J. Biol. Chem. 1983; 258: 4641-4647Abstract Full Text PDF PubMed Google Scholar) and found at 300 and 580 μg/ml in plasma of humans and mice, respectively (1Hynes R.O. Fibronectins. Springer-Verlag, New York1990Crossref Google Scholar, 7George E.L. Georges-Labouesse E.N. Patel-King R.S. Rayburn H. Hynes R.O. Development. 1993; 119: 1079-1091Crossref PubMed Google Scholar). pFN lacks both the EDA and EDB domains, whereas cellular FN is locally produced and deposited as insoluble fibrils in the extracellular matrix of tissues and contains these domains at variable proportions (1Hynes R.O. Fibronectins. Springer-Verlag, New York1990Crossref Google Scholar, 8ffrench-Constant C. Exp. Cell Res. 1995; 221: 261-271Crossref PubMed Scopus (172) Google Scholar, 9Kornblihtt A.R. Pesce C.G. Alonso C.R. Cramer P. Srebrow A. Werbajh S. Muro A.F. FASEB J. 1996; 10: 248-257Crossref PubMed Scopus (172) Google Scholar). Previous studies suggested that circulating pFN contributes to the extracellular matrix of tissues (10McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1983; 97: 466-472Crossref PubMed Scopus (201) Google Scholar, 11Oh E. Pierschbacher M. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3218-3221Crossref PubMed Scopus (181) Google Scholar) but the extent of the phenomenon has not been addressed. fibronectin plasma FN extra domain A type III homologies connecting segment wild type reverse transcription PCR. The levels of FN in plasma are critical for hemostasis, tissue repair, and susceptibility to infections. Depletion of pFN (liver-specific knockout of FN) results in increased brain injury after transient focal cerebral ischemia (12Sakai T. Johnson K.J. Murozono M. Sakai K. Magnuson M.A. Wieloch T. Cronberg T. Isshiki A. Erickson H.P. Fassler R. Nat. Med. 2001; 7: 324-330Crossref PubMed Scopus (283) Google Scholar), a delay in thrombus formation and decreased thrombus stability (13Ni H. Yuen P.S. Papalia J.M. Trevithick J.E. Sakai T. Fassler R. Hynes R.O. Wagner D.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2415-2419Crossref PubMed Scopus (190) Google Scholar), decreased angiogenesis (14Yi M. Sakai T. Fassler R. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11435-11438Crossref PubMed Scopus (64) Google Scholar), and increased susceptibility to bacterial infections (15Nyberg P. Sakai T. Cho K.H. Caparon M.G. Fassler R. Bjorck L. EMBO J. 2004; 23: 2166-2174Crossref PubMed Scopus (41) Google Scholar). Heterozygous null FN mice appear healthy and fertile (7George E.L. Georges-Labouesse E.N. Patel-King R.S. Rayburn H. Hynes R.O. Development. 1993; 119: 1079-1091Crossref PubMed Google Scholar) but show delayed thrombus growth in injured arterioles (16Matuskova J. Chauhan A.K. Cambien B. Astrof S. Dole V.S. Piffath C.L. Hynes R.O. Wagner D.D. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1391-1396Crossref PubMed Scopus (52) Google Scholar). Regrettably, the levels of FN present in the tissues of heterozygous null FN and in the pFN null mice have not been reported. We have previously shown that knock-in mice having constitutive inclusion of the EDA exon of the FN gene (EDA+/+ strain) had up to 70-80% reduction in the levels of plasma and tissue FN (17Muro A.F. Chauhan A.K. Gajovic S. Iaconcig A. Porro F. Stanta G. Baralle F.E. J. Cell Biol. 2003; 162: 149-160Crossref PubMed Scopus (246) Google Scholar). Taking advantage of the “floxed” EDA exon present in those mice, we generated liver-specific EDA-null mice (EDA+/+CRE) after crossing EDA+/+ animals with a transgenic strain expressing the CRE recombinase only in hepatocytes (18Kellendonk C. Opherk C. Anlag K. Schutz G. Tronche F. Genesis. 2000; 26: 151-153Crossref PubMed Scopus (198) Google Scholar). Consequently, hepatocytes of EDA+/+CRE mice, without the EDA exon, were able to produce and secrete pFN at normal levels. We show here that the levels of pFN were restored in those mice and also that the levels of tissue FN were similar to those observed in EDAWT/WT animals. These results showed a major flow of pFN into the extracellular matrix of tissues and suggest the importance of the pFN as an essential source of FN for the tissues. The presented results might have potential significance for understanding the contributions of pFN, and perhaps other plasma proteins, to cellular activities and in the formation of the extracellular matrix of tissues. Mice—The generation and genetic background of the mice devoid of regulated splicing at the EDA exon have been previously described (17Muro A.F. Chauhan A.K. Gajovic S. Iaconcig A. Porro F. Stanta G. Baralle F.E. J. Cell Biol. 2003; 162: 149-160Crossref PubMed Scopus (246) Google Scholar). EDA+/+ mice were mated with the transgenic strain Tg Alf pCRE mice, which have CRE recombinase under the control of the albumin promoter and enhancer (18Kellendonk C. Opherk C. Anlag K. Schutz G. Tronche F. Genesis. 2000; 26: 151-153Crossref PubMed Scopus (198) Google Scholar). EDA+/WT mice having the CRE recombinase were mated in order to obtain EDA+/+ mice with the CRE transgene (EDA+/+CRE). This strain expresses the CRE recombinase exclusively in hepatocytes (18Kellendonk C. Opherk C. Anlag K. Schutz G. Tronche F. Genesis. 2000; 26: 151-153Crossref PubMed Scopus (198) Google Scholar). The genotype of mice was determined by PCR from tail biopsies. RNA Preparation and Reverse Transcription (RT)-PCR Analysis—Total RNA was prepared from freshly extracted tissues and cells as described (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). The radioactive RT-PCR reactions were performed and quantified as previously described (20Chauhan A.K. Iaconcig A. Baralle F.E. Muro A.F. Gene. 2004; 324: 55-63Crossref PubMed Scopus (29) Google Scholar). Protein Extracts and Western Blot Analysis—Mice were anesthetized with 2.5% Avertin (300 μl/20 g mouse), and organs were perfused with 25 ml of cold phosphate-buffered saline through the left ventricle of the heart. Organs were immediately dissected and snap-frozen in liquid nitrogen. Organs were homogenized, and protein content was determined by Bradford protein assay (Bio-Rad). Identical amounts of protein sample were run on a 6% SDS-PAGE and analyzed by Western blot with polyclonal rabbit anti-FN antibody (50 μg of protein extract, 1:1500; Sigma), anti-EDA 3E2 monoclonal antibody (100 μg of protein extract, 1:300; Sigma), or anti β-tubulin monoclonal antibody (20 μg of protein extract, E7, 1:3000; Developmental Studies Hybridoma Bank, University of Iowa) as described (17Muro A.F. Chauhan A.K. Gajovic S. Iaconcig A. Porro F. Stanta G. Baralle F.E. J. Cell Biol. 2003; 162: 149-160Crossref PubMed Scopus (246) Google Scholar). Three animals per genotype were analyzed. To determine the efficiency of perfusion and elimination of plasma proteins in each of the organs analyzed, a Western blot analysis of 50 μg of protein extract was performed using an anti-mouse IgG antibody (1:2000; DAKO). Serial ECL expositions of the membranes were performed to determine the optimum linear range to quantify the signals. Films were scanned with the Versadoc (Bio-Rad) and quantified with the help of the Quantity One software package (Bio-Rad). Bile was collected by holding the gallbladder with forceps. Two μl of each sample were mixed with protein loading buffer (0.125 Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 0.002% bromphenol blue) and boiled for 5 min. Bile FN was analyzed by Western blot as described above. In Vivo Labeling of Hepatocytes—Hepatocytes were purified by the two-step collagenase perfusion method (21Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5225) Google Scholar), using Liver Digest Medium (Invitrogen) as described by the manufacturers. Hepatocytes were plated in rat tail collagen for 1 h, washed, and then incubated for 24 h with Met-Cys-free medium supplemented with 300 μCi/ml of [35S]Met/Cys (ProMix; Amersham Biosciences). The supernatant was collected, and a fraction was affinity-purified with gelatin-Sepharose as described (22Owens R.J. Baralle F.E. EMBO J. 1986; 5: 2825-2830Crossref PubMed Scopus (63) Google Scholar, 23Ruoslahti E. Hayman E.G. Pierschbacher M. Engvall E. Methods Enzymol. 1982; 82: Pt. A, 803-Pt. A, 831Crossref Scopus (470) Google Scholar). Southern Blot Analysis of Tissues—DNA was extracted from tissues, and 15 μg were digested with HindIII. The DNA was then run in an agarose gel and blotted onto Z-Probe membrane. A probe corresponding to the exon downstream to the EDA exon was used as described (17Muro A.F. Chauhan A.K. Gajovic S. Iaconcig A. Porro F. Stanta G. Baralle F.E. J. Cell Biol. 2003; 162: 149-160Crossref PubMed Scopus (246) Google Scholar). Immunohistochemistry of Tissue Sections—Organs were fixed in 4% formaldehyde and paraffin-embedded. 4-μm sections of each tissue were cut and incubated with affinity-purified polyclonal rabbit anti-FN antibody (1:200; Sigma). Then sections were incubated with biotinylated goat anti-rabbit IgG (5 μg/ml; VectaStain) followed by avidin-biotin-peroxidase mix (ABC Reagent; Vector Laboratories), 3,3′-diaminobenzidine peroxidase substrate (Vector Laboratories), and Gill's hematoxylin (Vector Laboratories). An AS LMD Leica microscope was used to visualize and photograph the sections. Hepatocytes of EDA+/+ Mice Have Normal Levels of Extracellular Matrix-FN but Do Not Secrete pFN—We have previously observed that mice having constitutive inclusion of the EDA exon of the FN gene (Fig. 1A) had a significant decrease of FN in plasma and in most tissues (17Muro A.F. Chauhan A.K. Gajovic S. Iaconcig A. Porro F. Stanta G. Baralle F.E. J. Cell Biol. 2003; 162: 149-160Crossref PubMed Scopus (246) Google Scholar, 20Chauhan A.K. Iaconcig A. Baralle F.E. Muro A.F. Gene. 2004; 324: 55-63Crossref PubMed Scopus (29) Google Scholar). Further characterization of pFN levels (embryo, young, and adult mice) from EDA+/+ mice showed very low amounts compared with EDAWT/WT and EDA-/- mice (Fig. 1, B and C). Embryos had 60% of the pFN levels in the control sample, whereas young and adult EDA+/+ mice showed a higher decrease in pFN levels. The decrease in pFN was due neither to lower levels in mRNA in tissues of EDA+/+ mice (17Muro A.F. Chauhan A.K. Gajovic S. Iaconcig A. Porro F. Stanta G. Baralle F.E. J. Cell Biol. 2003; 162: 149-160Crossref PubMed Scopus (246) Google Scholar) nor to a reduced FN production by EDA+/+ tissues, as FN secreted by EDA+/+ embryonic fibroblasts or adult heart fibroblasts was similar to that produced by the EDAWT/WT fibroblasts, excluding a general defect (17Muro A.F. Chauhan A.K. Gajovic S. Iaconcig A. Porro F. Stanta G. Baralle F.E. J. Cell Biol. 2003; 162: 149-160Crossref PubMed Scopus (246) Google Scholar). The specific degradation of FN in the EDA+/+ mice by proteases was ruled out by a series of experiments: 1) Metalloproteinase activity levels in plasma and tissues by gelatin zymography analysis were similar among EDAWT/WT, EDA+/+, and EDA-/- mice (supplemental Fig. S1, A and B); 2) [35S]Metlabeled fragments of FN containing or not containing the EDA exon were not differentially degraded when incubated with EDAWT/WT or EDA+/+ tissue extracts or plasma in the absence of protease inhibitors (supplemental Fig. S1, C and D); 3) no increase in FN degradation rate was observed after mixing protein extracts from EDA+/+ or EDAWT/WT liver with those originating from different organs or plasma from EDA+/+ mice in the absence of protease inhibitors (data not shown); 4) Western blot analysis of tissue extracts done with different sets of anti-FN polyclonal antibodies, run on 5-17% gradient gel, did not show any specific degradation products in EDA+/+ mice (data not shown). Because the hepatocytes are the source of pFN (5Owens M.R. Cimino C.D. Blood. 1982; 59: 1305-1309Crossref PubMed Google Scholar, 6Tamkun J.W. Hynes R.O. J. Biol. Chem. 1983; 258: 4641-4647Abstract Full Text PDF PubMed Google Scholar), we performed metabolic labeling of hepatocyte primary cultures from EDAWT/WT, EDA+/+, and EDA-/- livers followed by FN affinity purification of the conditioned medium to analyze pFN production. We observed a decreased secretion of pFN by the EDA+/+ hepatocytes, suggesting that the reduced levels in pFN in the EDA+/+ mice were the consequence of a defect in hepatocytes (Fig. 2, A and B). However, the FN amounts detected by Western blot in hepatocyte cell extracts were similar (Fig. 2C). Additionally, the decrease of pFN in EDA+/+ embryos (Fig. 1B) but not in embryonic tissues (17Muro A.F. Chauhan A.K. Gajovic S. Iaconcig A. Porro F. Stanta G. Baralle F.E. J. Cell Biol. 2003; 162: 149-160Crossref PubMed Scopus (246) Google Scholar) confirmed that the deficiency in FN secretion was limited only to EDA+/+ hepatocytes. These results showed that EDA+/+ hepatocytes were unable to secrete pFN in normal amounts. We hypothesized that the reduced levels of tissue FN could be due to the decreased supply of FN from plasma to tissues. Generation of Liver-specific EDA Null Mice—To determine the extent of FN flow into tissues we restored the capacity of hepatocytes to produce pFN not containing the EDA exon by cross-breeding EDA+/+ mice with a transgenic strain expressing the CRE recombinase only in hepatocytes (18Kellendonk C. Opherk C. Anlag K. Schutz G. Tronche F. Genesis. 2000; 26: 151-153Crossref PubMed Scopus (198) Google Scholar). The aim was to perform a tissue-specific deletion of the EDA exon without modifying the EDA+ allele in other cell types and tissues (Fig. 3, A and B). Southern blot analysis of different tissues from the EDA+/+ mice carrying the liver-specific CRE recombinase (EDA+/+CRE mice) showed CRE-mediated recombination only in the liver (Fig. 3C). The percentage of recombination in liver was ∼60-70%. Because hepatocytes constitute 60% of the total number of cells present in the liver (24Malarkey D.E. Johnson K. Ryan L. Boorman G. Maronpot R.R. Toxicol. Pathol. 2005; 33: 27-34Crossref PubMed Scopus (161) Google Scholar), one can consider that the percentage of recombination in hepatocytes was close to 100%. RT-PCR analysis confirmed the absence of the EDA exon in the FN mRNA both in liver and purified hepatocytes from EDA+/+CRE mice (Fig. 3D). FN Levels Are Restored in EDA+/+CRE Mice—Tissues and plasma from EDA+/+ mice expressing the hepatocyte-specific CRE recombinase were analyzed by Western blot with polyclonal anti-FN and anti-EDA-specific antibodies. As expected, pFN levels were completely restored in the EDA+/+CRE mice (Fig. 4A). Tissue extracts prepared from EDA+/+CRE mice contained amounts of FN similar to that found in EDAWT/WT mice (Fig. 4A). The amount of FN present in the extra-cellular matrix of tissues from EDA+/+CRE mice that was derived from plasma was ∼60% in testis and ∼40% in other tissues such as brain, heart, and lung (Fig. 4B). These results clearly indicate that an important proportion of the FN present in the extracellular matrix of adult tissues derives from plasma. Consequently, the amount of FN that is synthesized and deposited locally in tissues is much lower than believed. We then used the monoclonal antibody 3E2 to specifically detect the EDA-containing FN isoform. As expected, no EDA+FN was present in the plasma of EDAWT/WT mice. Additionally, we did not detect EDA+FN in the plasma of EDA+/+CRE mice, indicating a high efficiency of CRE-mediated recombination in hepatocytes. The EDA+FN isoform in liver was clearly visible only in the EDA+/+ samples (Fig. 5A, lanes 4-9), confirming again the Southern blot and RT-PCR data (Fig. 3, C and D). The absence of EDA+FN in the plasma of EDA+/+CRE mice also suggests that the FN flow is mainly from plasma to tissues and not vice versa (Fig. 5A, lanes 1-3). No differences were observed in the levels of EDA+FN in testis (Fig. 5, B and C) and in other tissues (data not shown) between the EDAWT/WT and EDA+/+CRE samples, suggesting that the observed differences in tissue FN among the different genotypes corresponded to EDA-FN incorporated from plasma into the extracellular matrix of tissues. To ensure that the detected levels of FN in tissues were not due to non-complete perfusion of the organs, we performed Western blot analysis of the plasma globulins that remained in each tissue after perfusion and normalized the protein load with the β-tubulin signal. Supplemental Fig. S2 shows that the remaining globulin levels after tissue perfusion were ∼10% of the levels seen in the non-perfused organs. Because most plasma proteins were washed out from the perfused organs, the differences of FN levels among the different strains were not due to residual contamination of pFN. Immunostaining of Tissue Sections Confirmed the Decrease of FN Levels in EDA+/+ Tissues and the Recovery in Tissues of EDA+/+CRE Mice—The above results were confirmed by immunohistochemical analysis of tissue sections. Similar levels of FN-specific signal were detected in the sinusoids of liver samples prepared from all three genotypes (Fig. 6, A-C, black arrows). In the brain sections of EDAWT/WT and EDA+/+CRE mice a stronger FN-specific signal, probably associated with the cell surface of glial cells (25Price J. Hynes R.O. J. Neurosci. 1985; 5: 2205-2211Crossref PubMed Google Scholar, 26Tom V.J. Doller C.M. Malouf A.T. Silver J. J. Neurosci. 2004; 24: 9282-9290Crossref PubMed Scopus (155) Google Scholar, 27Yang Z. Suzuki R. Daniels S.B. Brunquell C.B. Sala C.J. Nishiyama A. J. Neurosci. 2006; 26: 3829-3839Crossref PubMed Scopus (105) Google Scholar), was observed when compared with the EDA+/+ mice (Fig. 6E, white arrows). Similarly, in testis tissue sections, a stronger FN-specific signal was observed in the basement membrane of seminiferous tubules and interstitial regions of EDAWT/WT and EDA+/+CRE mice compared with EDA+/+ samples (Fig. 6H, white arrows). However, we did not observe intracellular accumulation of FN in the tissue sections of EDA+/+ mice by immunohistochemical analysis (Fig. 6, B, E, and H) or in primary culture of hepatocytes either by FN immunofluorescence (supplemental Fig. 3) or by metabolic labeling (Fig. 2D). Gross histology of tissue samples from all three genotypes was similar, suggesting that the FN synthesized locally is sufficient to maintain the normal tissue architecture. These results indicate that there was a flow of FN from plasma to the extracellular matrix of tissues and plasma is an important source of tissue FN. Our data demonstrate an important and novel role for plasma proteins, in particular that of fibronectin, in the formation of the extracellular matrix of tissues and, probably, in the modulation of cellular activities in tissues. The concept that there is FN flow from plasma into tissues or extracellular matrix of cells has been known for a long time. Addition or injection of soluble FN into the culture medium of cells or into the plasma of mice, respectively, resulted in the incorporation of FN into the extracellular matrix (10McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1983; 97: 466-472Crossref PubMed Scopus (201) Google Scholar, 11Oh E. Pierschbacher M. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3218-3221Crossref PubMed Scopus (181) Google Scholar, 28Peters D.M. Portz L.M. Fullenwider J. Mosher D.F. J. Cell Biol. 1990; 111: 249-256Crossref PubMed Scopus (71) Google Scholar, 29Sottile J. Hocking D.C. Swiatek P.J. J. Cell Sci. 1998; 111: 2933-2943Crossref PubMed Google Scholar, 30Bae E. Sakai T. Mosher D.F. J. Biol. Chem. 2004; 279: 35749-35759Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). However, the magnitude of the contribution of pFN to the extracellular matrix was not possible to address with either model. In the present report we have shown that in some tissues up to 60% of the fibronectin present in the extracellular matrix could be plasma-derived. Sakai et al. (12Sakai T. Johnson K.J. Murozono M. Sakai K. Magnuson M.A. Wieloch T. Cronberg T. Isshiki A. Erickson H.P. Fassler R. Nat. Med. 2001; 7: 324-330Crossref PubMed Scopus (283) Google Scholar) have recently shown that pFN supports neuronal survival and reduces brain injury following transient focal cerebral ischemia, suggesting the incorporation of pFN into the injured brain. Our results confirmed and extended their observations to non-injured tissues as we showed pFN incorporation into most normal organs, including brain. In fact, we are also demonstrating that this is a general mechanism that occurs in most normal tissues, and we suggest that other plasma proteins could also become incorporated into the extracellular matrix of tissues, modulating cellular activities. Because FN is found both in blood and bile fluids, secretion of FN by hepatocytes seems not to be polarized as proposed for endothelial cells (31Kowalczyk A.P. Tulloh R.H. McKeown-Longo P.J. Blood. 1990; 75: 2335-2342Crossref PubMed Google Scholar). Furthermore, we also observed a decrease in FN levels in the bile fluid of EDA+/+ mice (data not shown), suggesting the absence of an EDA-dependent polarization of FN secretion in hepatocytes, as postulated for airway epithelial cells (32Wang A. Cohen D.S. Palmer E. Sheppard D. J. Biol. Chem. 1991; 266: 15598-15601Abstract Full Text PDF PubMed Google Scholar). The low levels of soluble FN in the plasma of EDA+/+ mice and intermediate levels in EDA+/WT and EDA+/- mice (data not shown) point toward the existence of a mechanism, analogous to that observed for the secretion of the IIICS variants (33Schwarzbauer J.E. Spencer C.S. Wilson C.L. J. Cell Biol. 1989; 109: 3445-3453Crossref PubMed Scopus (70) Google Scholar), that detects the EDA domain during the secretory pathway of pFN from hepatocytes and prevents the release of EDA+FN into the bloodstream. The “defective” EDA+/EDA+ pFN dimers might be formed but their transit through the pFN secretory pathway might be slower compared with EDA-/EDA- dimers. However, immunostaining of tissue sections, immunofluorescence of primary culture of hepatocytes, or metabolic labeling of hepatocytes did not reveal any intracellular accumulation of FN in the liver of EDA+/+ mice, suggesting that the putative “misfolded” dimers are quickly degraded. We observed that the amount of pFN supplied to the extracellular matrix of tissues varied among the different organs analyzed, suggesting that there might be equilibrium between the amount of FN locally produced by the tissues and the availability of cellular receptors to incorporate FN from the plasma pool. To conclude, the presented results might have potential significance for understanding the contributions of pFN, and perhaps other plasma proteins, to cellular activities and in the formation of the extracellular matrix of tissues, as we have shown that a major fraction of tissue FN is plasma-derived. Because plasma provides approximately an equal amount of FN as the tissue itself, we suggest that plasma should be considered an important source of FN for tissues. Furthermore, hepatocytes have normal levels of FN in the extracellular matrix but do not secrete soluble EDA+FN, suggesting the existence of separate secretory pathways for soluble and fibrillar FN. We thank Marcello Raspa and Günther Schütz for providing the Tg Alf pCRE mice, Meghan Walsh for critical reading of the manuscript and for comments regarding the use of English, and Giancarlo Lunazzi, Mauro Sturnega, and Stefano Artico for help in animal handling. Download .pdf (1.81 MB) Help with pdf files" @default.
• W2134839779 created "2016-06-24" @default.
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• W2134839779 date "2007-09-01" @default.
• W2134839779 modified "2023-10-12" @default.
• W2134839779 title "A Major Fraction of Fibronectin Present in the Extracellular Matrix of Tissues Is Plasma-derived" @default.
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• W2134839779 doi "https://doi.org/10.1074/jbc.m611315200" @default.
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