Proline recycling during collagen metabolism as determined by concurrent 18O2- and 3H-labeling

In a previous study where rat skin collagen was labeled with ¹⁸O in the hydroxyl group of the collagen hydroxyproline we noticed that the decay rate of this label was much faster than had been observed when the skin collagen hydroxyproline was labeled with ³H in the prolyl ring. In this study a rat was labeled concurrently with [¹⁸O₂] and [³H] proline and the rate of decline of both labels was determined in rat skin collagen hydroxyproline. After correction for growth dilution of the skin collagen the [¹⁸O] hydroxyproline was found to have a half-life of 27 days while the [³H] hydroxyproline had a half-life of 53 days. The decay rate of the [¹⁸O] hydroxyproline represents the true turnover rate of collagen since there is no possibility of recycling this label. Hence, the difference between this and the [³H] hydroxyproline decay rate is due to recycling of l-[³H] proline into new collagen. The efficiency of recycling of proline from catabolized collagen into new collagen was about 93%.     

Inhibition by oestrogen of collagen breakdown in the involuting rat uterus

1. Both the post-partum involution of the rat uterus and the rapid breakdown of collagen that accompanies it are extensively inhibited by oestrogenic hormones. In the normal rat, 85% of the uterine collagen is degraded within 4 days after parturition; in rats treated with 100μg. of 17β-oestradiol/day, only 35% of uterine collagen is broken down in the same period. 2. Similar effects are produced by diethylstilboestrol if the dose is increased tenfold. 3. Collagen breakdown is inhibited to a greater extent than is the loss of wet weight by oestradiol but not by diethylstilboestrol. 4. The oestrogens appear to act by blocking the breakdown of collagen. There is a greatly decreased concentration of free hydroxyproline in the uterus of treated animals. 5. Acid hydrolase concentrations (β-glucuronidase, β-galactosidase, cathepsin D and acid phosphatase) in the uterus are decreased by oestrogen treatment compared with controls, but the total amounts of these enzymes in the uterus are somewhat elevated. Oestrogens do not appear to inhibit collagen breakdown by altering the concentration and total amount of acid hydrolases.     

Hydroxyproline reaction with free radicals generated during benzoyl peroxide catalytic decomposition of carbon tetrachloride Structure of reaction products formed

Summary Benzoyl peroxide catalytic decomposition of carbon tetrachloride in a model system produces trichloromethyl and trichloromethylperoxyl free radicals. These radicals are also produced by CCl₄ bioactivation in liver and are considered to be responsible for the deleterious effects of this hepatotoxin. In this study, it is attempted to learn about how the .CCl₃ and CCl₃O₂. tend to react with hydroxyproline in a model system. Hydroxyproline was selected because of its role in collagen metabolism. During the interaction of both radicals with hydroxyproline a total of 16 reaction products were isolated and identified by gas chromatographic-mass spectrometric analysis. All of them were hydroxyproline analogs, no single one contained C from CCl₄ and only three contained chlorine. Consequently, most adducts would be missed in experiments where formation of reaction products are studied by formation of¹⁴C or³⁶Cl labeled adducts (e.g. covalent binding studies used by toxicologists). If similar hydroxyproline analog reaction products were observed during CCl₄ intoxication it might be reasonably expected that they interfered with collagen metabolism and participate in cirrhogenic effects of CCl₄ on the liver.     

Glucose deficiency reduces collagen synthesis in breast cancer MCF7 cells

We decided to study the effect of glucose deprivation on collagen metabolism in MCF7 cells. The incorporation of [³H]‐proline into collagenase‐sensitive and hydroxyproline‐containing proteins was used as an index of collagen synthesis, whereas pulse—chase technique was employed to evaluate the degradation of newly synthesized proteins. The MCF7 cells incubated in high glucose medium synthesized detectable amounts of collagenous proteins. Most of them were found in the cell layer. The shortage of glucose resulted in about 30% reduction in collagen synthesis. The pulse—chase experiments demonstrated that proportionally less collagen was degraded in cultures incubated in low‐glucose than in high‐glucose media.     

β1 Integrins Mediate Chondrocyte Interaction with Type I Collagen, Type II Collagen, and Fibronectin

Chondrocytes isolated from the cephalic region of sterna from 14-day-old chick embryos used β1 integrins and required either Mg²⁺ or Mn²⁺ for attachment to plates coated with type I collagen, type II collagen, and fibronectin. β1 integrin was concentrated in adhesion plaques of the chondrocytes plated on type I collagen, type II collagen, and fibronectin substrates. Chondrocytes expressed at least 3 α-subunits, including α3, α5, and putative α2. α5, but not α3, had a higher molecular weight in chondrocytes than in fibroblasts. Levels of α3 and α5 were about 25-30% of that in fibroblasts. When the chondrocytes were cultured in the presence of ascorbate in suspension, the cells aggregated into clusters. This aggregation was dependent on β1 integrin and type II collagen.     

Actinidain-hydrolyzed Type I Collagen Reveals a Crucial Amino Acid Sequence in Fibril Formation

We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of α1 and α2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized by poor fibrillogenesis. Then, the N- and C-terminal sequences of chicken type I collagen hydrolyzed by actinidain or pepsin were determined by Edman degradation and de novo sequence analysis with matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry, respectively. In the C-telopeptide region of the α1 chain, pepsin cleaved between Asp¹⁰³⁵ and Phe¹⁰³⁶, and actinidain between Gly¹⁰³² and Gly¹⁰³³. Thus, the actinidain-hydrolyzed α1 chain is shorter at the C terminus by three residues, Gly¹⁰³³, Phe¹⁰³⁴, and Asp¹⁰³⁵. In the α2 chain, both proteases cleaved between Glu¹⁰³⁰ and Val¹⁰³¹. We demonstrated that a synthetic nonapeptide mimicking the α1 C-terminal sequence including GFD weakly inhibited the self-assembly of pepsin-hydrolyzed collagen, whereas it remarkably accelerated that of actinidain-hydrolyzed collagen. We conclude that the specific GFD sequence of the C-telopeptide of the α1 chain plays a crucial role in stipulating collagen suprastructure and in subsequent fibril formation.     

Role of PTPα in the Destruction of Periodontal Connective Tissues

IL-1β contributes to connective tissue destruction in part by up-regulating stromelysin-1 (MMP-3), which in fibroblasts is a focal adhesion-dependent process. Protein tyrosine phosphatase-α (PTPα) is enriched in and regulates the formation of focal adhesions, but the role of PTPα in connective tissue destruction is not defined. We first examined destruction of periodontal connective tissues in adult PTPα^+/+ and PTPα^−/− mice subjected to ligature-induced periodontitis, which increases the levels of multiple cytokines, including IL-1β. Three weeks after ligation, maxillae were processed for morphometry, micro-computed tomography and histomorphometry. Compared with unligated controls, there was ∼1.5–3 times greater bone loss as well as 3-fold reduction of the thickness of the gingival lamina propria and 20-fold reduction of the amount of collagen fibers in WT than PTPα^−/− mice. Immunohistochemical staining of periodontal tissue showed elevated expression of MMP-3 at ligated sites. Second, to examine mechanisms by which PTPα may regulate matrix degradation, human MMP arrays were used to screen conditioned media from human gingival fibroblasts treated with vehicle, IL-1β or TNFα. Although MMP-3 was upregulated by both cytokines, only IL-1β stimulated ERK activation in human gingival fibroblasts plated on fibronectin. TIRF microscopy and immunoblotting analyses of cells depleted of PTPα activity with the use of various mutated constructs or with siRNA or PTPα^KO and matched wild type fibroblasts were plated on fibronectin to enable focal adhesion formation and stimulated with IL-1β. These data showed that the catalytic and adaptor functions of PTPα were required for IL-1β-induced focal adhesion formation, ERK activation and MMP-3 release. We conclude that inflammation-induced connective tissue degradation involving fibroblasts requires functionally active PTPα and in part is mediated by IL-1β signaling through focal adhesions.     

Genetic Analysis of the Role of Protein Kinase Cθ in Platelet Function and Thrombus Formation

Background

PKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ^−/− T cells exhibit reduced activation and PKCθ^−/− mice are resistant to autoimmune disease, making PKCθ an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCθ positively regulates outside-in signalling through integrin α_IIbβ₃ in platelets, the role of PKCθ in GPVI-dependent signalling and functional activation of platelets has not been assessed.

Methodology/Principal Findings

In the present study we assessed static adhesion, cell spreading, granule secretion, integrin α_IIbβ₃ activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ^−/− platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ^−/− platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of α_IIbβ₃ activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s⁻¹) was enhanced.

Conclusions/Significance

These data suggest that PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and α_IIbβ₃ activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors.     

Inhibition of Hb Binding to GP1bα Abrogates Hb-Mediated Thrombus Formation on Immobilized VWF and Collagen under Physiological Shear Stress

BackgroundReports including our own describe that intravascular hemolysis increases the risk of thrombosis in hemolytic disorders. Our recent study shows that plasma Hb concentrations correlate directly with platelet activation in patients with paroxysmal nocturnal hemoglobinuria (PNH). The binding of Hb to glycoprotein1bα (GP1bα) increases platelet activation. A peptide AA1-50, designed from N-terminal amino acid sequence of GP1bα significantly inhibits the Hb binding to GP1bα as well as Hb-induced platelet activation. This study further examined if the Hb-mediated platelet activation plays any significant role in thrombus formation on subendothelium matrix under physiological flow shear stresses and the inhibition of Hb-platelet interaction can abrogate the above effects of Hb.

Methods and Results

Study performed thrombus formation assay in vitro by perfusing whole blood over immobilized VWF or collagen type I in presence of Hb under shear stresses simulating arterial or venous flow. The Hb concentrations ranging from 5 to 10 μM, commonly observed level in plasma of the hemolytic patients including PNH, dose-dependently increased thrombus formation on immobilized VWF under higher shear stress of 25 dyne/cm², but not at 5 dyne/cm². The above Hb concentrations also increased thrombus formation on immobilized collagen under both shear stresses of 5 and 25 dyne/cm². The peptide AA1-50 abrogated invariably the above effects of Hb on thrombus formation.

Conclusions and Significance

This study therefore indicates that the Hb-induced platelet activation plays a crucial role in thrombus formation on immobilized VWF or collagen under physiological flow shear stresses. Thus suggesting a probable role of this mechanism in facilitating thrombosis under hemolytic conditions.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.     

Fibroblast α11β1 Integrin Regulates Tensional Homeostasis in Fibroblast/A549 Carcinoma Heterospheroids

We have previously shown that fibroblast expression of α11β1 integrin stimulates A549 carcinoma cell growth in a xenograft tumor model. To understand the molecular mechanisms whereby a collagen receptor on fibroblast can regulate tumor growth we have used a 3D heterospheroid system composed of A549 tumor cells and fibroblasts without (α11^+/+) or with a deletion (α11^-/-) in integrin α11 gene. Our data show that α11^-/-/A549 spheroids are larger than α11^+/+/A549 spheroids, and that A549 cell number, cell migration and cell invasion in a collagen I gel are decreased in α11^-/-/A549 spheroids. Gene expression profiling of differentially expressed genes in fibroblast/A549 spheroids identified CXCL5 as one molecule down-regulated in A549 cells in the absence of α11 on the fibroblasts. Blocking CXCL5 function with the CXCR2 inhibitor SB225002 reduced cell proliferation and cell migration of A549 cells within spheroids, demonstrating that the fibroblast integrin α11β1 in a 3D heterospheroid context affects carcinoma cell growth and invasion by stimulating autocrine secretion of CXCL5. We furthermore suggest that fibroblast α11β1 in fibroblast/A549 spheroids regulates interstitial fluid pressure by compacting the collagen matrix, in turn implying a role for stromal collagen receptors in regulating tensional hemostasis in tumors. In summary, blocking stromal α11β1 integrin function might thus be a stroma-targeted therapeutic strategy to increase the efficacy of chemotherapy.     

The timecourse of collagen cross-linking

The incorporation of [³H] proline into all major denaturation subunits of acid-extractable skin collagen was followed in vivo over a period of 30 days in young male rats. The radioactivity was incorporated into the α₁ and α₂ subunits at an equal rate. It was then transferred to the β subunits, the ratio of specific activity of α:β becoming unity at about 17 days. No transfer of radioactivity from β to γ collagen was seen during the 30 days of the experiment indicating that β units do not cross-link further to γ during this time. A separate group of rats made lathyritic with β-aminopropionitrile showed a similar rate of collagen cross-linking as reflected by the return of α:β ratios to normal values within about 15 days following the cessation of β-aminopropionitrile administration. In the normal rats radioactive proline was incorporated very rapidly into a fraction of collagen eluted from CM-cellulose with 0.5 M NaCl following the salt gradient. It is proposed that this fraction may represent a fibrogenesis nucleation factor.     

α2β1 Integrin,GPVI Receptor,and Common FcRγ Chain on Mouse Platelets Mediate Distinct Responses to Collagen in Models of Thrombosis

Objective

Platelets express the α2β1 integrin and the glycoprotein VI (GPVI)/FcRγ complex, both collagen receptors. Understanding platelet-collagen receptor function has been enhanced through use of genetically modified mouse models. Previous studies of GPVI/FcRγ-mediated collagen-induced platelet activation were perfomed with mice in which the FcRγ subunit was genetically deleted (FcRγ^−/−) or the complex was depleted. The development of α2β1^−/− and GPVI^−/− mice permits side-by-side comparison to address contributions of these collagen receptors in vivo and in vitro.

Approach and Results

To understand the different roles played by the α2β1 integrin, the GPVI receptor or FcRγ subunit in collagen-stimulated hemostasis and thrombosis, we compared α2β1^−/−, FcRγ^−/−, and GPVI^−/− mice in models of endothelial injury and intravascular thrombosis in vivo and their platelets in collagen-stimulated activation in vitro. We demonstrate that both the α2β1 integrin and the GPVI receptor, but not the FcRγ subunit influence carotid artery occlusion in vivo. In contrast, the GPVI receptor and the FcRγ chain, but not the α2β1 integrin, play similar roles in intravascular thrombosis in response to soluble Type I collagen. FcRγ^−/− platelets showed less attenuation of tyrosine phosphorylation of several proteins including RhoGDI when compared to GPVI^−/− and wild type platelets. The difference between FcRγ^−/− and GPVI^−/− platelet phosphotyrosine levels correlated with the in vivo thrombosis findings.

Conclusion

Our data demonstrate that genetic deletion of GPVI receptor, FcRγ chain, or the α2β1 integrin changes the thrombotic potentials of these platelets to collagen dependent on the stimulus mechanism. The data suggest that the FcRγ chain may provide a dominant negative effect through modulating signaling pathways in platelets involving several tyrosine phosphorylated proteins such as RhoGDI. In addition, these findings suggest a more complex signaling network downstream of the platelet collagen receptors than previously appreciated.     

Efficient Inhibition of Collagen-Induced Platelet Activation and Adhesion by LAIR-2, a Soluble Ig-Like Receptor Family Member

LAIR-1 (Leukocyte Associated Ig-like Receptor -1) is a collagen receptor that functions as an inhibitory receptor on immune cells. It has a soluble family member, LAIR-2, that also binds collagen and can interfere with LAIR-1/collagen interactions. Collagen is a main initiator for platelet adhesion and aggregation. Here, we explored the potential of soluble LAIR proteins to inhibit thrombus formation in vitro. LAIR-2/Fc but not LAIR-1/Fc inhibited collagen-induced platelet aggregation. In addition, LAIR-2/Fc also interfered with platelet adhesion to collagen at low shear rate (300 s⁻¹; IC₅₀ = 18 µg/ml) and high shear rate (1500 s⁻¹; IC₅₀ = 30 µg/ml). Additional experiments revealed that LAIR-2/Fc leaves interactions between collagen and α2β1 unaffected, but efficiently prevents binding of collagen to Glycoprotein VI and von Willebrand factor. Thus, LAIR-2/Fc has the capacity to interfere with platelet-collagen interactions mediated by Glycoprotein VI and the VWF/Glycoprotein Ib axis.     

The regulatory influence of prostaglandins on protein synthesis in the human non pregnant cervix

Cervical connective tissue was obtained from non pregnant fertile women undergoing hysterectomy. Tissue specimens were mechanically chopped into 1 mm thick slices which were preincubated in Krebs-Ringer bicarbonate buffer containing PGE₂ or PGF_2α (300 ng/ml). After 60 min the slices were transferred into fresh buffer with the corresponding PG-concentration and ³H-labelled proline or hydroxyproline. Following incubation (60 min) the protein bound radioactivity was determined and related either to the dry weight or to the protein content of each slice. The two methods used did not show any qualitative differences. The experiments showed that PG:s had a marked effect on protein synthesis in the cervical tissue. During the follicular phase incubation with both types of PG:s was followed by a decreased incorporation indicating decreased net synthesis of collagen while there was an increased incorporation and hence increased synthesis in the luteal phase. There was no significant influence on the distribution of the model amino acid ¹⁴C-AIB in the presence of PG:s neither in the follicular nor in the luteal phase. The present data point to an acute effect of PGE₂ and PGF_2α on cervical collagen metabolism and indicate furthermore that the process is steroid hormone dependent. It is concluded that these substances may exert their effect by modulating the incorporation of amino acids into protein.     

Dihydrocytochalasin B Enhances Transforming Growth Factor-β-Induced Reexpression of the Differentiated Chondrocyte Phenotype without Stimulation of Collagen Synthesis

Rabbit articular chondrocytes were treated with retinoic acid (RA) to eliminate the differentiated phenotype marked by the synthesis of type II collagen and high levels of proteoglycan. Exposure of such cells to transforming growth factor-β1 (TGF-β1) in secondary culture under serum-free and RA-free, defined conditions led to reexpression of the differentiated phenotype. The microfilament modifying drug, dihydrocytochalasin B (DHCB), enhanced the effectiveness of TGF-β1 and produced a threefold stimulation of type II collagen reexpression (measured by 2-D CNBr peptide mapping) at 0.3 ng/ml TGF-β1 without altering total collagen synthesis. Type II collagen reexpression was maximal from 1 to 5 ng/ml TGF-β1, with or without DHCB. The effect of DHCB on proteoglycan synthesis was maximal at 1 ng/ml TGF-β1. At this dose TGF-β alone produced no increase in ³⁵ SO₄ incorporation, while simultaneous treatment with DHCB caused a sevenfold stimulation of proteoglycan synthesis. DHCB-independent stimulation of proteoglycan reexpression occurred between 5 and 15 ng/ml TGF-β1. In contrast, TGF-β1-dependent stimulation of proteoglycan synthesis in differentiated chondrocytes in primary monolayer culture was not substantially affected by DHCB. The collagen data suggest that TGF-β1 utilizes separate pathways to control phenotypic change and collagen (matrix) synthesis. Microfilament modification by DHCB selectively enhances the effectiveness of the TGF-β1-dependent signaling pathway that controls reexpression of the differentiated phenotype.     

Synthesis and Secretion of Hydroxyproline-containing Macromolecules in Carrots: II. In vivo Conversion of Peptidyl Proline to Peptidyl Hydroxyproline

The chelator α,α′-dipyridyl prevents the formation of protein-bound hydroxyproline without affecting protein synthesis as measured by the incorporation of ¹⁴C-proline. The inhibitory effect can be overcome by exogenous ferrous ions. Neither α,α′-dipyridyl nor Fe²⁺ interferes in any other known way with the synthesis and secretion of the hydroxyproline-rich proteins normally found in the cell wall. This reversal by Fe²⁺ of the inhibition of proline hydroxylation by α,α′-dipyridyl is used for a study in vivo of the hydroxylation reaction. This reaction has a temperature coefficient of 2.2, suggesting that it is an enzymatic process. Reversal by Fe²⁺ of the α,α′-dipyridyl inhibition can occur after protein synthesis has been arrested with cycloheximide, indicating that peptidyl proline is the substrate of the hydroxylation reaction. Hydroxylation occurs in the cytoplasm, and not in the cell wall, and only for a limited time after the incorporation of proline into the polypeptide chain. This suggests a spatial separation of the enzyme and the substrate.     

Enhancing Integrin α1 Inserted (I) Domain Affinity to Ligand Potentiates Integrin α1β1-mediated Down-regulation of Collagen Synthesis

Integrin α1β1 binding to collagen IV, which is mediated by the α1-inserted (I) domain, down-regulates collagen synthesis. When unligated, a salt bridge between Arg²⁸⁷ and Glu³¹⁷ is thought to keep this domain in a low affinity conformation. Ligand binding opens the salt bridge leading to a high-affinity conformation. How modulating integrin α1β1 affinity alters collagen homeostasis is unknown. To address this question, we utilized a thermolysin-derived product of the α1α2α1 network of collagen IV (α1α2α1(IV) truncated protomer) that selectively binds integrin α1β1. We show that an E317A substitution enhanced binding to the truncated protomer, consistent with a previous finding that this substitution eliminates the salt bridge. Surprisingly, we show that an R287A substitution did not alter binding, whereas R287E/E317R substitutions enhanced binding to the truncated protomer. NMR spectroscopy and molecular modeling suggested that eliminating the Glu³¹⁷ negative charge is sufficient to induce a conformational change toward the open state. Thus, the role played by Glu³¹⁷ is largely independent of the salt bridge. We further show that cells expressing E317A or R287E/E317R substitutions have enhanced down-regulation of collagen IV synthesis, which is mediated by the ERK/MAPK pathway. In conclusion, we have demonstrated that modulating the affinity of the extracellular α1 I domain to collagen IV enhances outside-in signaling by potentiating ERK activation and enhancing the down-regulation of collagen synthesis.     

Silencing CD36 gene expression results in the inhibition of latent-TGF-β1 activation and suppression of silica-induced lung fibrosis in the rat

Background

The biologically active form of transforming growth factor-β1 (TGF-β1) plays a key role in the development of lung fibrosis. CD36 is involved in the transformation of latent TGF-β1 (L-TGF-β1) to active TGF-β1. To clarify the role of CD36 in the development of silica-induced lung fibrosis, a rat silicosis model was used to observe both the inhibition of L-TGF-β1 activation and the antifibrotic effect obtained by lentiviral vector silencing of CD36 expression.

Methods

The rat silicosis model was induced by intratracheal injection of 10 mg silica per rat and CD36 expression was silenced by administration of a lentiviral vector (Lv-shCD36). The inhibition of L-TGF-β1 activation was examined using a CCL-64 mink lung epithelial growth inhibition assay, while determination of hydroxyproline content along with pathological and immunohistochemical examinations were used for observation of the inhibition of silica-induced lung fibrosis.

Results

The lentiviral vector (Lv-shCD36) silenced expression of CD36 in alveolar macrophages (AMs) obtained from bronchoalveolar lavage fluid (BALF) and the activation of L-TGF-β1 in the BALF was inhibited by Lv-shCD36. The hydroxyproline content of silica+Lv-shCD36 treated groups was significantly lower than in other experimental groups. The degree of fibrosis in the silica+Lv-shCD36-treated groups was less than observed in other experimental groups. The expression of collagen I and III in the silica+Lv-shCD36-treated group was significantly lower than in the other experimental groups.

Conclusion

These results indicate that silencing expression of CD36 can result in the inhibition of L-TGF-β1 activation in a rat silicosis model, thus further preventing the development of silica-induced lung fibrosis.