Fibronectin Biosynthesis Research Articles

Characterization and immunocytochemical localization of actin and fibronectin in haemocytes of the mussel Mytilus galloprovincialis.

Cell-extracellular matrix interactions are recognized to be important for human leucocyte functions, including chemotaxis and phagocytosis. These activities depend on a reorganization of the microfilament actin (F-actin) promoted by fibronectin, one of the major components of extracellular matrices. Although invertebrate haemocytes are, in many aspects, similar to the human granulocyte-monocyte-macrophage cell lineage, actin and fibronectin have not been well studied in these cells. Consequently, the characterization and structural organization of actin and fibronectin in mussel (Mytilus galloprovincialis) haemocytes was investigated using Western blotting analysis, indirect immunofluorescence and immunoelectron microscopy. Actin was immunocharacterized by an anti-total actin monoclonal antibody. Fibronectin was immunocharacterized by an autologous polyclonal antiserum directed against the protein of mussel haemolymph. Actin was mainly localized along the peripheral cytoplasm of the haemocyte. The distribution of the F-actin microfilaments was assayed with Rhodamine-labelled phalloidin. F-actin was associated mainly with stress-fibres of spreading haemocytes and with microspikes at the adhesion sites. The labelling by the anti-fibronectin antiserum of the haemocyte rough endoplasmic reticulum vesicles, revealed by immunoelectron microscopy, suggests that these cells are involved in fibronectin biosynthesis. Gold particles were also present along the outer surfaces of the cell plasma membrane and its protrusions. Mussel fibronectin was localized immunohistochemically at the adhesion sites and in the extracellular matrix fibrils. The relationships between fibronectin and the actin cytoskeleton in Mytilus galloprovincialis haemocytes are discussed.

Comparative studies between freshly isolated and spontaneously immortalized bovine granulosa cells: Protein secretion, steroid metabolism, and responsiveness to growth factors

A bovine granulosa cell line (BGC-1) has been obtained by spontaneous immortalization of primary cultures. BGC-1 cells have retained some characteristics of primary cultures, such as the hormonal regulation of fibronectin biosynthesis. In the present study we have compared BGC-1 cells and primary cultures of bovine granulosa cells in terms of protein secretion, steroid metabolism, and mitogenic responses to growth factors. The pattern of protein secretion in BGC-1 cells was qualitatively similar to that of primary cultures. The main differences were a higher proportion of fibronectin and the relative amounts of several other unidentified proteins. Progesterone levels in BGC-1 cultures were undetectable. When BGC-1 cells and primary cultures were incubated with [3H]-pregnenolone, the former showed a lower conversion rate to progesterone. In contrast, the conversion rate of [3H]-progesterone to 5 alpha-reduced metabolites was markedly increased in BGC-1 cells. We also examined the effects of epidermal growth factor (EGF), insulin like growth factor-I (IGF-I), and transforming growth factor-beta (TGF-beta) on DNA synthesis under serum-free conditions. Both primary cultures and BGC-1 cells exhibited a stimulatory response to EGF and IGF-I on [3H]-thymidine incorporation. Neither BGC-1 cells nor primary cultures showed a significant response to TGF-beta when added alone. However, in the presence of a combination of EGF and IGF-I, TGF-beta displayed an inhibitory effect on primary cultures while it stimulated DNA synthesis in BGC-1 cells even further. The addition of conditioned medium from BGC-1 cells (BGC-1-CM) stimulated DNA synthesis on primary cultures to a greater extent than the addition of conditioned medium from primary cultures. These results suggest that BGC-1 cells may be a useful model to study the regulation of granulosa cell function during the period previous to the preovulatory stage of follicular development. The differential responses of the immortalized cells to growth regulators may offer some clues on the mechanisms that control cell proliferation in normal tissues.

Biosynthesis of basement membrane molecules by salivary adenoid cystic carcinoma cells: an immunofluorescence and confocal microscopic study.

The biosynthesis of basement membrane molecules and fibronectin was studied in vitro in the two different human cell systems (ACC2 and ACC3) established from adenoid cystic carcinomas (ACC) of the salivary gland using immunofluorescence and confocal microscopy. When cells were attached and spread on dishes, fine granular immunofluorescence for type IV collagen, laminin, heparan sulphate proteoglycan, entactin, and fibronectin first appeared diffusely in the cytoplasm, and then changed in aggregation of coarse granules in the perinuclear area. With formation of colonies, these signals were present in the extracellular space, initially in the basal aspect of attached cells and consequently in the lateral intercellular space. After the cells formed a confluent monolayer, extracellular signals started to decrease in inverse proportion to the reappearance of intracellular ones. The results indicate that the parenchymal cells of ACC synthesize these five extracellular matrix molecules, secrete them into the extracellular milieu and remodel the extracellular deposits. It is suggested that the characteristic stromal architecture of ACC, represented by stromal pseudocysts, results from their own secretion of the basement membrane molecules and fibronectin.

Transcriptional Repression of Fibronectin Gene Expression in v-src Transformation

V-src-dependent and -independent alterations in the steady-state content, rates of synthesis, and turnover of fibronectin protein and mRNA were identified using rat fibroblasts which are temperature sensitive for p60v-src activity. Activation of p60v-src caused a fivefold reduction in the rate of fibronectin biosynthesis. The v-src-dependent decrease in fibronectin biosynthesis resulted from a similar reduction in the steady-state content of fibronectin mRNA. This change was reversible and required more than 24 h, implying an indirect effect of p60v-src on fibronectin gene expression. The rate of fibronectin mRNA turnover and pattern of alternative splicing were unchanged following p60v-src activation, indicating that these regulatory steps are insensitive to v-src transformation. A v-src-specific reduction of at least threefold was measured for the rate of fibronectin gene transcription, and gene transfer studies using fibronectin promoter-CAT reporter genes indicated that transcriptional repression occurs at the level of initiation. When p60v-src was inactive, CAT reporter genes controlled by 4.9 or 3.2 kb of the rat fibronectin promoter exhibited relatively increased CAT activity (∼twofold) compared to another CAT reporter construction controlled by only 880 bp of the fibronectin promoter. In contrast, CAT activity was relatively reduced (∼twofold) for the reporter constructions containing 4.9 or 3.2 kb of the promoter when p60v-src was active. These findings indicate that the distal portion of the fibronectin promoter contains a v-src-sensitive element(s) which mediates a decrease in the rate of fibronectin transcription initiation by negative control.

Increased Cell-Substrate Adhesion Accompanies Conditional Reversion to the Normal Phenotype in Ras-Oncogene-Transformed NIH-3T3 Cells

We recently reported (1991, Mol. Cell Biol. 11, 3699-3710) that depletion of c-myc protein by myc antisense sequences in ras-transformed NIH-3T3 cells reverses several of the malignant characteristics of these cells. These include transformed morphology, growth in soft agar, and ability to form tumors in athymic mice. In the present study we examined the same cells for in vitro adhesive behavior. Cells depleted of c-myc protein by antisense transfection showed no change in attachment to fibronectin-coated dishes as compared to ras-transformed NIH-3T3 cells but had greatly increased resistance to trypsin/EDTA-mediated release from the substratum after attachment. In concomitant studies, the cells were examined for fibronectin biosynthesis and cell surface fibronectin. There was no overall change in fibronectin biosynthesis in the c-myc antisense transfected cells as compared to the ras-transformed NIH-3T3. However, immunofluorescence staining revealed increased amount of surface fibronectin associated with the antisense c-myc-expressing transfectants. Taken together, these data indicate that the conditional reacquisition of the nonmalignant phenotype in ras-transformed NIH-3T3 cells by selected depletion of c-myc protein is associated with an increase in cell-substrate adhesion. This, in turn, is associated with an increase in surface fibronectin.

A novel mechanism of Ha-ras oncogene action: regulation of fibronectin mRNA levels by a nuclear posttranscriptional event.

Although loss of cell surface fibronectin (FN) is a hallmark of many oncogenically transformed cells, the mechanisms responsible for this phenomenon remain poorly understood. The present study utilized the nontumorigenic human osteosarcoma cell line TE-85 to investigate the effects of induced Ha-ras oncogene expression on FN biosynthesis. TE-85 cells were stably transfected with metallothionein-Ha-ras fusion genes, and the effects of metal-induced ras expression on FN biosynthesis were determined. Induction of the ras oncogene, but not proto-oncogene, was accompanied by a decrease in total FN mRNA and protein levels. Transfection experiments indicated that these oncogene effects were not due to reduced FN promoter activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. However, in this study the down-regulation of FN was identified as a nuclear event. A component of the ras effect was due to a mechanism affecting accumulation of processed nuclear FN RNA. Mechanisms that would generate such an effect include altered RNA processing and altered stability of the processed message in the nucleus. There was no effect of ras on FN mRNA poly(A) tail length or site of polyadenylation. There was also no evidence for altered splicing at the ED-B domain of FN mRNA. This demonstration of nuclear posttranscriptional down-regulation of FN by the Ha-ras oncogene identifies a new level at which ras oncoproteins can regulate gene expression and thus contribute to development of the malignant phenotype.

A novel mechanism of Ha-ras oncogene action: regulation of fibronectin mRNA levels by a nuclear posttranscriptional event.

Although loss of cell surface fibronectin (FN) is a hallmark of many oncogenically transformed cells, the mechanisms responsible for this phenomenon remain poorly understood. The present study utilized the nontumorigenic human osteosarcoma cell line TE-85 to investigate the effects of induced Ha-ras oncogene expression on FN biosynthesis. TE-85 cells were stably transfected with metallothionein-Ha-ras fusion genes, and the effects of metal-induced ras expression on FN biosynthesis were determined. Induction of the ras oncogene, but not proto-oncogene, was accompanied by a decrease in total FN mRNA and protein levels. Transfection experiments indicated that these oncogene effects were not due to reduced FN promoter activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. However, in this study the down-regulation of FN was identified as a nuclear event. A component of the ras effect was due to a mechanism affecting accumulation of processed nuclear FN RNA. Mechanisms that would generate such an effect include altered RNA processing and altered stability of the processed message in the nucleus. There was no effect of ras on FN mRNA poly(A) tail length or site of polyadenylation. There was also no evidence for altered splicing at the ED-B domain of FN mRNA. This demonstration of nuclear posttranscriptional down-regulation of FN by the Ha-ras oncogene identifies a new level at which ras oncoproteins can regulate gene expression and thus contribute to development of the malignant phenotype.

Fibronectin Biosynthesis in Endothelial Regeneration after Intimal Injury

In this experiment, we sought to identify the major fibronectin (FN) synthesizing cells, the source of FN production, and the role of FN in intimal regeneration. In rats in which vascular endothelial denudation had been induced, serial morphologic changes after intimal injury were studied by light and electron microscopic examination using immunohistochemical techniques and in situ hybridization. At 2 weeks after intimal injury, regenerated endothelial cells had pleomorphic cytoplasm and loose cellular junctions. Immunohistochemically, factor VIII-related antigen was localized in the regenerated endothelial cells, and immunoreaction products of FN were increased in the thickened neointima. Ultrastructurally, FN was localized in the proliferated endoplasmic reticulum of regenerated endothelial cells in the intima, while the α5 subunit of α5β1 integrin, one of the fibronectin receptors, was localized in the plasma membrane and increased endoplasmic reticulum of regenerated endothelial cells. FN mRNA was localized in a large number of regenerated endothelial cells (87.2 and 89.8%) by in situ hybridization at 2 and 4 weeks after intimal injury. These findings indicate that FN may mainly contribute to endothelial cell functions such as spreading and adhesion in the regenerative stage.

Angiotensin II stimulates human fetal mesangial cell proliferation and fibronectin biosynthesis by binding to AT1 receptors

The renin-angiotensin system is activated during vascular development and injury. Furthermore, angiotensin II (Ang II) is a comitogen for fetal mesangial cells in vitro and it may be important in vascular smooth cell growth in disease states. Since fibronectin is an important extracellular matrix protein for vascular development and it too is overexpressed in the mesangium of diseased glomeruli, we explored the interrelationship of fibronectin and Ang II in fetal mesangial cell growth. In human fetal kidney, Ang II type 2 receptors (AT2) were detected in abundance by ex vivo autoradiography. When mesangial cells were isolated from fetal kidney and grown in culture, Ang II type 1 receptors (AT1) were also detected. To explore the mitogenic properties Ang II and fibronectin as well as the effects of Ang II on fibronectin metabolism, studies were performed in vitro, isolated from the potentially confounding variables of hemodynamic influence and circulating growth factors and cytokines. In vitro, mesangial cells expressed a single class of AT1 receptors that were not altered by growth on various substrates. Ang II (10(-7) M) significantly increased thymidine incorporation by confluent human fetal mesangial cells (twofold). When subconfluent, Ang II-stimulated proliferation was greater (fourfold). Ang II significantly increased cell-associated and secreted fibronectin as determined by immunoprecipitation at concentrations that also stimulate mitogenesis. Both of these Ang II-mediated responses were inhibited by the AT1 receptor antagonist DuP-753 (10(-5) M) but not by AT2 receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of fetal fibronectin production in human chorion cells

Our purpose was to evaluate the role of human chorion in production of fetal fibronectin and to assess the regulation of fetal fibronectin production by inflammatory products and cytokines. Human chorion cells were grown in culture, and at confluence cells were incubated with and without lipopolysaccharide, interleukin-1 beta, tumor necrosis factor-alpha, and acetylsalicylic acid. Fetal fibronectin production was measured in the supernatants by means of enzyme-linked immunosorbent assay. Production of fetal fibronectin was increased by lipopolysaccharide, interleukin-1 beta, and tumor necrosis factor-alpha. Acetylsalicylic acid had no effect on fetal fibronectin biosynthesis. Fetal fibronectin production by human chorion cells in vitro is stimulated by inflammatory products and mediators, which are considered to be important in the initiation of some cases of preterm labor.

Role of stromal cells and macrophages in fibronectin biosynthesis and matrix assembly in human long-term marrow cultures

Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.

Role of Stromal Cells and Macrophages in Fibronectin Biosynthesis and Matrix Assembly in Human Long-Term Marrow Cultures

AbstractFibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunopreci-pitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth musclelike stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa + fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L20ri —, were also able to synthesize the EDa + fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa —, EDb — fibronectin variant, clearly distinct from the EDa + variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa +, EDb — fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa +, EDb — fibronectin instead of EDa —, EDb — soluble fibronectin, as found in human plasma.

Biomarkers of connective tissue aging: biosynthesis of fibronectin, collagen type III, and elastase.

Annals of the New York Academy of SciencesVolume 673, Issue 1 p. 16-22 Biomarkers of Connective Tissue Aging: Biosynthesis of Fibronectin, Collagen Type III, and Elastase J. LABAT-ROBERT, J. LABAT-ROBERT Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this authorP. KERN, P. KERN Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this authorL. ROBERT, L. ROBERT Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this author J. LABAT-ROBERT, J. LABAT-ROBERT Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this authorP. KERN, P. KERN Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this authorL. ROBERT, L. ROBERT Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this author First published: December 1992 https://doi.org/10.1111/j.1749-6632.1992.tb27431.xCitations: 29AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume673, Issue1Physiopathological Processes of Aging: Towards a Multicausal InterpretationDecember 1992Pages 16-22 RelatedInformation

Fibronectin Gene Transcription Is Enhanced in Abnormal Wound Healing

Abnormal wound healing in susceptible individuals can result in the formation of keloids that have an elevated content of extracellular matrix material compared to normal scars. Keloid-derived fibroblasts exhibit as much as a four-fold increase in the rate of fibronectin biosynthesis compared to fibroblasts from normal dermis and normal scars. Altered biosynthesis is due to an increase in the steady-state level of fibronectin mRNA, and in this investigation we have identified the level of fibronectin gene expression that is responsible for this increase. The rate of fibronectin gene transcription was found to be increased as much as threefold in keloid fibroblasts when compared to normal fibroblasts. Other possible changes that could account for the elevated level of fibronectin mRNA in keloids, such as increased copy number of the fibronectin gene or decreased turnover of fibronectin mRNA were also examined. The possibility of altered gene dosage was eliminated because chromosome content, G-banding patterns, and fibronectin gene content of keloid fibroblasts were all found to be normal. Analysis of fibronectin mRNA degradation revealed a half-life of approximately 13 h, and the residual fibronectin mRNA was observed to remain full length during this time period in both keloid and normal fibroblasts. Thus, altered degradation of fibronectin mRNA is unlikely to contribute to overproduction of fibronectin in keloids. Increased translational competence of fibronectin mRNA in keloids was also eliminated as a contributing factor because fibronectin mRNA remaining after one half-life were equally available for translation in both cell types. Although stimulation of transcription may not entirely account for the increase in fibronectin biosynthesis in keloids, this mechanism is best able to account for the majority of the change.

Keloid Fibroblasts Exhibit an Altered Response to TGF-β

Treatment with transforming growth factor beta 1 (TGF-beta 1) results in stimulation of total protein synthesis in normal dermal fibroblasts but not in keloid fibroblasts, suggesting that the TGF-beta regulatory program is altered in keloid fibroblasts. However, both keloid and normal fibroblasts treated with TGF-beta 1 exhibit accelerated fibronectin biosynthesis, indicating that keloid cells can respond to TGF-beta 1. In the absence of serum, the TGF-beta 1-induced increase in fibronectin biosynthesis occurs more rapidly in keloid fibroblasts, also suggesting modification of this regulatory pathway. The TGF-beta 1-mediated increase in keloid fibronectin production is independent of the steroid regulatory pathway for fibronectin, which accelerates synthesis by means of a post-transcriptional mechanism. Thus, TGF-beta 1 stimulation of fibronectin production in keloid cells is likely to involve a transcriptional mechanism and keloid overproduction of extracellular matrix components may be due to an inherent modification of the TGF-beta regulatory program.

UVA- and UVB-induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice

The modifications induced in hairless mouse skin by chronic UV irradiation were investigated. Skin explant cultures were used to study UVA- and UVB-induced changes occurring in interstitial collagen (type I and type III) and fibronectin biosynthesis. To study the long-term effects, albino hairless mice were irradiated with UVA radiation alone from two sources with different spectral qualities or with UVB. UVA and UVB radiation produced a significant increase in the ratio of type III to type I collagen (more than 100% for UVA-irradiated skin and about 60% for UVB-irradiated skin) accompanied by a significantly increased fibronectin biosynthesis (50% or more in all irradiated groups). Irradiation with either UVA or UVB alone had no significant effect on the total collagen synthesis and resulted in only a slight decrease in the total collagen content of the skin determined as hydroxyproline. This decrease was significant only in the case of the group irradiated with UVA (xenon) (decrease of 25%, expressed as micrograms of hydroxyproline per milligram wet weight). A significant decrease in collagen hydroxylation (expressed as radioactive hydroxyproline/radioactive hydroxyproline plus proline in neosynthesized collagen) was observed of about 50% in skin irradiated with UVA (xenon) but not in UVB-treated skin. Several of the above modifications (increased fibronectin biosynthesis, increased collagen type III to type I ratio) correspond to the modifications observed during the aging of non-irradiated hairless mice. Therefore it appears that UV irradiation accelerates the modifications of extracellular matrix biosynthesis observed during aging.

In vivo evaluation of photoprotection against chronic ultraviolet-A irradiation by a new sunscreen Mexoryl SX.

In a previous study on the hairless mouse it was shown that sub-erythemal doses of pure UV-A enhanced the numerous changes normally observed during chronological aging. A new sunscreen (a bis-benzylidene campho sulfonic acid derivative) has been synthesized in our research laboratory (lambda max: 345 nm, epsilon: 47,000). Its photoprotective properties against UV-A induced damages were assessed in our mouse model. Three month old albino hairless mice were exposed for 1 y to suberythemal doses (35 J/cm2) of UV-A obtained from a xenon source filtered through a WG 345 filter. One group of animals was exposed untreated, the other received a formulation containing 5% of the sunscreen prior to irradiation. At the end of the study the cutaneous properties of protected mice were compared to those of unprotected animals and to 3 and 15 month old unirradiated controls. We found that the visible changes induced by UV-A irradiation were mainly sagging and wrinkling. Histological and electron microscopic alterations consisted of hyperkeratosis, increased density of elastic fibers with alteration of fiber orientation and increased glycosaminoglycan deposits. Biochemical changes consisted of decreases in total collagen and collagen hydroxylation and increases in both collagen III/I + III ratio and fibronectin biosynthesis. All these changes were reduced or abolished by the sunscreen.

Dexamethasone modulates α2-macroglobulin and apolipoprotein E gene expression in cultured rat liver fat-storing (Ito) cells

Fat-storing (Ito) cells are perisinusoidal liver cells thought to play a central role in vitamin A metabolism and fibrongenesis. Glucocorticoids have been shown to be beneficial in the treatment of certain types of liver diseases by delaying the development of cirrhosis. To study the regulatory effects of dexamethasone on Ito cell gene expression, Ito cells were isolated from normal rat liver and primary cultures were established. The effect of dexamethasone on the synthesis of alpha 2-macroglobulin, apolipoprotein E, fibronectin and actin was examined. Protein synthesis was studied both at the protein level and at the RNA level by means of biosynthetic labeling, immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Northern blot analysis of total RNA. After exposure to dexamethasone for 20 hr, alpha 2-macroglobulin protein synthesis was increased threefold, whereas apolipoprotein E expression was decreased 80%. Biosynthesis of fibronectin remained unaffected by hormone treatment. The dexamethasone effect became detectable 5 hr after beginning the exposure. Deinduction kinetic experiments showed that the glucocorticoid effect was detectable more than 12 hr after the replacement of the dexamethasone-containing culture medium by medium without the hormone. Corresponding to the data obtained at the protein level, dexamethasone increased the steady-state levels of alpha 2-macroglobulin-specific messenger RNA and reduced apolipoprotein E-specific transcripts, whereas fibronectin and actin messenger mRNA remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibronectin biosynthesis in the rat aorta in vitro. Changes due to experimental hypertension.

This study was undertaken to determine if changes in fibronectin biosynthesis accompany the phenotypic changes that occur in aortic tissue following experimental hypertension. An in vitro procedure was developed to measure fibronectin synthesis in aortic rings obtained from normotensive or hypertensive rats. There was a three to sixfold increase in fibronectin biosynthesis by aortic rings taken from rats treated with deoxycorticosterone/salt for 7 and 21 d, the change being more pronounced at 21 d. In contrast, there was no major change at either time point in net incorporation into total protein. Studies comparing fibronectin biosynthesis in aortic rings from Wistar rats and spontaneously hypertensive rats at ages between 10 and 40 wk showed increased fibronectin biosynthesis in older animals of both strains, but only slight differences between strains. Studies using rats infused with angiotensin II showed a correlation between blood pressure elevation and increased aortic fibronectin biosynthesis. Western blot analysis of aortic extracts showed that the fibronectin content was increased in the hypertensive models. The in vitro procedure for measuring fibronectin biosynthesis appears to provide a reliable reflection of in vivo changes in fibronectin expression, and the methodology could prove useful for studying the factors influencing protein expression in vascular tissue.

Enhanced expression of fibronectin by cmd/cmd chondrocytes and its modulation by exogenously added proteoglycan

Cartilage matrix deficiency (cmd/cmd) in mice is a genetic disorder associated with the failure of chondrocytes to synthesize the core protein of cartilage proteoglycan monomer (cartilage-PG). Immunohistochemical and biochemical analyses revealed enhanced accumulation of fibronectin in culture of cmd/cmd chondrocytes as well as in mutant cartilage tissue in vivo. Purified cartilage-PG, when added exogenously to a culture of cmd/cmd chondrocytes, caused a reduction in abnormal accumulation of fibronectin over several subsequent days of culturing. Using a fibronectin cDNA probe, we showed that, on the basis of total RNA, the fibronectin mRNA level was four or eight times higher in cmd/cmd chondrocytes than in the normal. The level of fibronectin mRNA in cmd/cmd cells was lowered by culturing the cells in the presence of added cartilage-PG. These findings suggest that the abnormal accumulation of fibronectin in the mutant cell culture is primarily due to elevation of fibronectin mRNA level, and that cartilage-PG in the extracellular matrix may affect the regulation of fibronectin biosynthesis at the steady-state level of mRNA.