Comparison of the Late Triassic carbonate platform evolution and Lofer cyclicity in the Transdanubian Range, Hungary and Pelagonian Zone, Greece

Abstract For comparative studies of Upper Triassic cyclic platform carbonates, the Transdanubian Range (Hungary) and the Pelagonian Zone (Greece) were chosen. Paleogeographically they represent two distant segments of the passive margin of the Neotethys Ocean. During the Late Triassic, on this wide margin a very extensive tropical carbonate platform domain was developed, referred to as the Dachstein-type carbonate platform system. The Transdanubian Range (TR) represents a segment of a continent-encroaching platform system, whereas the Pelagonian-Subpelagonian Zone (PG) may have been a large isolated platform, surrounded by deep-water basins. The discussed Upper Triassic thick platform carbonates (Fődolomit/Hauptdolomit Formation and Dachstein Limestone in the TR, and Pantokrator Formation in the PG) are made up of cyclically arranged facies deposited under similar environmental conditions in the interior zones of carbonate platforms. Three characteristic major facies types can be distinguished: shallow subtidal-lagoonal, intertidal and supratidal-pedogenic, which correspond to the three typical lithofacies (members C, B and A) of Fischer's (1964) Lofer-cycle. The cycles are usually bounded by discontinuity surfaces related to subaerial exposure and pedogenic alteration. The meter-scale (Lofer) cyclicity is predominant throughout the successions. However, various stacking patterns including symmetric complete, truncated, incomplete, and condensed cycles or even alternating peritidal and subtidal facies without any disconformity are recognized in both areas studied. Pervasive or partial early diagenetic dolomitization affected some parts of the cyclic successions in both areas. However, age-dependence of the early dolomitization was clearly demonstrated only in the TR, where the older part of the platform carbonate succession was subject to pervasive dolomitization, whereas the younger part is non-dolomitized and there is a transitional unit between them. This trend is attributed to the climate changing from semiarid to more humid. The Upper Triassic platform carbonates of the TR and PG show strikingly similar features concerning the litho- and biofacies, the stacking pattern and the thickness of the elementary cycles, despite their distant and different paleogeographic setting within the western Neotethys realm. This suggests a eustatic signal, i.e. the cyclic deposition was essentially controlled by orbitally-forced eustatic sea-level changes, although the contribution of autocyclic mechanisms cannot be excluded either. Definite signatures of subaerial exposure (karstic features and vadose meteoric diagenesis) at and below the cycle boundaries also support allocyclic control. In the northeastern part of the TR the carbonate platform was drowned at the Triassic-Jurassic boundary, whereas platform conditions persisted until the end of the Hettangian in the southwestern part. However, the Hettangian part of the succession is characterized by non-cyclic subtidal limestone, implying an upward-deepening trend. In contrast, in the PG the platform conditions continued until early to middle Liassic, and the Liassic succession is typified by well-developed pedogenic features, suggesting long-lasting subaerial exposure intervals, i.e. an upward-shallowing trend

Central Role of SREBP-2 in the Pathogenesis of Osteoarthritis

Background: Recent studies have implied that osteoarthritis (OA) is a metabolic disease linked to deregulation of genes involved in lipid metabolism and cholesterol efflux. Sterol Regulatory Element Binding Proteins (SREBPs) are transcription factors regulating lipid metabolism with so far no association with OA. Our aim was to test the hypothesis that SREBP-2, a gene that plays a key role in cholesterol homeostasis, is crucially involved in OA pathogenesis and to identify possible mechanisms of action. Methodology/Principal Findings: We performed a genetic association analysis using a cohort of 1,410 Greek OA patients and healthy controls and found significant association between single nucleotide polymorphism (SNP) 1784G>C in SREBP-2 gene and OA development. Moreover, the above SNP was functionally active, as normal chondrocytes’ transfection with SREBP-2-G/C plasmid resulted in interleukin-1β and metalloproteinase-13 (MMP-13) upregulation. We also evaluated SREBP-2, its target gene 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR), phospho-phosphoinositide3-kinase (PI3K), phospho-Akt, integrin-alphaV (ITGAV) and transforming growth factor-$\beta$ (TGF-$\beta$) mRNA and protein expression levels in osteoarthritic and normal chondrocytes and found that they were all significantly elevated in OA chondrocytes. To test whether TGF-$\beta$ alone can induce SREBP-2, we treated normal chondrocytes with TGF-$\beta$ and found significant upregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13. We also showed that TGF-$\beta$ activated aggrecan (ACAN) in chondrocytes only through Smad3, which interacts with SREBP-2. Finally, we examined the effect of an integrin inhibitor, cyclo-RGDFV peptide, on osteoarthritic chondrocytes, and found that it resulted in significant upregulation of ACAN and downregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13 expression levels. Conclusions/Significance: We demonstrated, for the first time, the association of SREBP-2 with OA pathogenesis and provided evidence on the molecular mechanism involved. We suggest that TGF-$\beta$ induces SREBP-2 pathway activation through ITGAV and PI3K playing a key role in OA and that integrin blockage may be a potential molecular target for OA treatment

Investigation of genes involved in lipid metabolism in Osteoarthritis and their regulatory mechanisms: in vitro and in vivo study

Purpose: The purpose of the present thesis is the investigation of the molecular basis of lipid metabolism in osteoarthritic chondrocytes through evaluation of the expression of genes that control cholesterol homeostasis, such as Sterol Regulatory Element Protein 2 (SREBP–2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the implicated mechanisms controlling their activation, and the regulation of cholesterol efflux related genes using microRNAs. In addition, we developed an experimental animal model of OA in order to highlight potential therapeutic molecules for inhibiting disease progression in vivo.Methods: Expression of genes involved in lipid metabolism was evaluated using real-time PCR and Western blot. We treated chondrocytes with TGF-β1 and integrin inhibitor and investigated the signal transduction pathway responsible for the activation of lipid metabolism genes. We also treated chondrocytes with miR-33a and anti-miR-33a and investigated their role on cholesterol synthesis and on the expression of cholesterol efflux related genes. Finally, we developed animal model of OA induced by anterior cruciate ligament transection in rabbits.Results: We observed that SREBP–2, HMGCR, integrin aV, TGF–β1, phosphor-PI3k and phosphor-Akt expression levels were significantly upregulated in osteoarthritic chondrocytes compared to normal. Treatment of normal chondrocytes with TGF–β1 resulted in SREBP–2, HMGCR, phospho–PI3K and MMP–13 upregulation. The use of the pharmacological inhibitor of TGF–β receptor resulted in downregulation of SREBP–2 expression while integrin blockage resulted in dramatic reduction of SREBP–2, HMGCR, phospho–PI3K and MMP–13 expression levels accompanied by elevated levels of ACAN. We also found that miR–33a expression levels were significantly elevated in osteoarthritic chondrocytes compared to normal, in accordance to SREBP–2’s upregulation and that miR–33a expression was increased in a time–dependent manner after TGF–β1 activation. Treatment of normal chondrocytes with miR–33a resulted in significant reduction of ABCA1 and ApoA1 mRNA expression levels, accompanied by increased levels of MMP–13. Introduction of antisense oligonucleotides directed against miR–33a, resulted in strongly increased ABCA1 and ApoA1 expression levels, while it also caused reduced expression of MMP–13. Our in vivo results showed that SREBP–2 expression levels were significantly elevated in rabbit OA chondrocytes compared to normal. Intra-articular administration of TGF–β receptor inhibitor resulted in a statistically significant reduction in the expression levels of SREBP–2, COLX and MMP–13, whereas COL2A1 and ACAN were significantly increased.Conclusion: Our novel findings suggest that SREBP–2 is crucially involved in OA pathogenesis and provided evidence for its TGF–β1–induced activation through ITGAV/PI3K/Akt pathway, pointing towards the use of integrin inhibitors as possible molecular targets for osteoarthritis treatment. Moreover, we provide novel evidence for the implication of miR–33a, a SREBP–2 intronic microRNA, in OA pathogenesis, identifying it as a dual regulator of cholesterol synthesis and cholesterol efflux–related genes in OA chondrocytes, suggesting its potential use as a novel target for the amelioration of OA phenotype. We finally found that TGF–β inhibition in vivo may be a new potential therapeutic approach for the treatment of the disease.Σκοπός: Αντικείμενο της διατριβής αποτέλεσε η διερεύνηση του μεταβολικού φαινοτύπου της ΟΑ μέσω μελέτης της έκφρασης γονιδίων που ενέχονται στο μεταβολισμό των λιπιδίων, όπως το SREBΡ–2 και το HMGCR, των μοριακών σηματοδοτικών μονοπατιών που ελέγχουν τη δράση τους, καθώς και η ρύθμιση της έκφρασης γονιδίων που ενέχονται στη σύνθεση και στην έξοδο της χοληστερόλης με τη χρήση microRNAs. Επιπλέον, δημιουργήθηκε πειραματικό ζωικό μοντέλο ΟΑ για την ανάδειξη πιθανών θεραπευτικών μορίων της νόσου in vivo. Μεθοδολογία: Ο προσδιορισμός της έκφρασης γονιδίων που συμμετέχουν στο μεταβολισμό των λιπιδίων έγινε με τις μεθόδους της real-time PCR και του Western blot. Η διερεύνηση του σηματοδοτικού μονοπατιού που ευθύνεται για την ενεργοποίηση της έκφρασής τους έγινε με τη χορήγηση TGF-β1 και αναστολέα των ιντεγκρινών στα χονδροκύτταρα. Επιπλέον, έγινε χορήγηση του miR-33a καθώς και του αναστολέα του anti-miR-33a στα χονδροκύτταρα προκειμένου να διερευνηθεί ο ρόλος του στη ρύθμιση της σύνθεσης της χοληστερόλης αλλά και στην έκφραση των γονιδίων που ενέχονται στην έξοδο της χοληστερόλης από τα χονδροκύτταρα. Τέλος, δημιουργήθηκε πειραματικό μοντέλο ΟΑ επαγόμενο με διατομή του πρόσθιου χιαστού συνδέσμου σε κουνέλια.Αποτελέσματα: Παρατηρήθηκε αυξημένη έκφραση του SREBP–2, του HMGCR, της ιντεγκρίνης aV, του TGF–β1 και των phosphor-PI3k και phosphor-Akt στα ΟΑ χονδροκύτταρα συγκριτικά με τα φυσιολογικά. Η επαγωγή χονδροκυττάρων με TGF–β1 προκάλεσε φωσφορυλίωση των PI3k και Akt και αύξηση της έκφρασης των γονιδίων SREBP–2 και HMGCR. Η αναστολή του TGF–β1 επέφερε μείωση των επιπέδων έκφρασης του SREBP–2, ενώ αναστολή της έκφρασης των ιντεγκρινών επέφερε σημαντική μείωση της έκφρασης των SREBP–2 και HMGCR, καθώς και της ενεργής μορφής της κινάσης PI3K. Στη συνέχεια, διαπιστώσαμε αυξημένη έκφραση του miR–33a στα ΟΑ χονδροκύτταρα συγκριτικά με τα φυσιολογικά και επαγωγή της έκφρασής του από τον TGF–β1, ενώ η χρήση αναστολέα του miR–33a σε επαγόμενα με TGF–β1 χονδροκύτταρα οδήγησε σε μείωση της φωσφορυλίωσης της πρωτεΐνης Akt. Επιπλέον, η χορήγηση του miR–33a στα φυσιολογικά χονδροκύτταρα επέφερε μείωση της έκφρασης των γονιδίων ABCA1 και ApoA1 και ταυτόχρονη αύξηση των επιπέδων της MMP–13, γεγονός που αντιστράφηκε μετά τη χρήση αναστολέα του miR–33a στα ΟΑ χονδροκύτταρα. Στο in vivo πειραματικό τμήμα της διατριβής διαπιστώσαμε στατιστικά σημαντική αύξηση των επιπέδων έκφρασης του SREBP–2 στα ΟΑ χονδροκύτταρα συγκριτικά με τα φυσιολογικά, ενώ η ενδοαρθρική χορήγηση αναστολέα των υποδοχέων του TGF–β προκάλεσε στατιστικά σημαντική μείωση των επιπέδων έκφρασης του SREBP–2, του COLX και της MMP–13, με ταυτόχρονη αύξηση των μεταγραφικών επιπέδων των αναβολικών μορίων COL2A1 και ACAN.Συμπεράσματα: Αναδείχτηκε για πρώτη φορά η εμπλοκή του SREBP–2 και του HMGCR στα ΟΑ χονδροκύτταρα καθώς και ο σηματοδοτικός μηχανισμός ρύθμισής τους μέσω των κινασών PI3K και Akt, ως αποτέλεσμα της ενεργοποίησης του TGF–β σηματοδοτικού μονοπατιού και της αυξημένης έκφρασης της ιντεγκρίνης aV στα ΟΑ χονδροκύτταρα. Επιπλέον, η αναστολή του miR–33a, η οποία επάγει την αυξημένη έκφραση γονιδίων που ευθύνονται για την έξοδο της περίσσειας χοληστερόλης από τα ΟΑ χονδροκύτταρα, αναδεικνύεται ως πιθανός θεραπευτικός μοριακός στόχος για την αναστολή της εξέλιξης της ΟΑ. Επίσης, η αναστολή του TGF–β in vivo, αναδεικνύεται ως μία νέα θεραπευτική προσέγγιση για την αντιμετώπιση της νόσου

Alleles of 1784G>C (rs2228314) polymorphism in SREBP-2 gene.

<p>Alleles of 1784G>C (rs2228314) polymorphism in SREBP-2 gene.</p

Effect of TGF-β receptor inhibitor (SB-431542) on SREBP-2 expression and ITGAV expression in normal and osteoarthritic chondrocytes.

<p>(<b>A</b>) Western blot showing SREBP-2 protein expression levels in normal chondrocytes treated with 10 ng/ml TGF-β, 10 ng/ml TGF-β plus 1 µΜ SB-431542, and 10 ng/ml TGF-β plus 10 µΜ SB-431542 for 6 h. GAPDH was used as loading control. (<b>B</b>) MMP-13 and COL2A1 mRNA expression levels in normal chondrocytes treated with 10 ng/ml TGF-β, 10 ng/ml TGF-β plus 1 µΜ SB-431542, and 10 ng/ml TGF-β plus 10 µΜ SB-431542 for 6 h, normalized to GAPDH. *p<0.05, NS: non significant (<b>C</b>) ITGAV mRNA expression in normal and osteoarthritic chondrocytes. GAPDH was used for normalization of the Real Time PCR data. All experiments were performed in duplicate. Data are expressed as mean. *p<0.05 (<b>D</b>) A representative western blot showing increased protein levels of ITGAV in osteoarthritic chondrocytes compared to normal, where β-actin was used as loading control.</p

Schematic representation of the molecular pathway activated in osteoarthritic chondrocytes where the involvement of 1784G>C polymorphism is evident.

<p>Briefly, in OA, ITGAV and TGF-β upregulation leads to phospho-PI3K and phospho-Akt activation which in turn cause overexpression of SREBP-2 and its target gene HMGCR accompanied by increased levels of MMP-13. Interestingly, chondrocytes expressing the SREBP-2 G/C genotype bind stronger to Smad3 leading to ACAN downregulation, contributing thus to the osteoarthritic phenotype. Furthermore, inhibition of integrins in OA, leads to SREBP-2, HMGCR and MMP-13 downregulation with subsequent elevation of ACAN levels suggesting integrin blockage as a potential molecular target for OA treatment.</p

Transfection of chondrocytes with plasmids and interactions between SREBP-2 and Smad3.

<p>(<b>A</b>) Primary osteoarthritic chondrocytes were transfected by electroporation with SREBP-2 WT and SREBP-2 G/C plasmids. 24 h post transfection we performed immunoprecipitation for SREBP-2 and tested the interaction with Smad3. (<b>B</b>) Normal chondrocytes were treated with TGF-β (3, 10 ng/ml) and Smad3 activity was assessed by luciferase activity (GACA) 12-luc plasmid, 12 h post transfection. (<b>C</b>) Western blot analysis showing Smad3 protein expression levels in normal chondrocytes treated for 24 h with siRNA control (100 nM) or siRNA against Smad3 (100 nM). GAPDH was used as loading control. (<b>D</b>) Normal chondrocytes were treated with TGF-β (10 ng/ml) in the presence of siRNA control (100 nM) or siRNA against Smad3 (100 nM). (<b>E</b>) Normal chondrocytes were treated with TGF-β (10 ng/ml) and transfected with SREBP-2 WT or SREBP-2 G/C plasmids and ACAN mRNA expression was tested by real-time PCR. (<b>F</b>) SREBP-2 G/C overexpression in normal chondrocytes up-regulated MMP-13 and IL-1β in higher levels in comparison to SREBP-2 WT over-expression (48 h post transfection).</p

Effect of integrin blockage by cyclo-RGDFV peptide (RGD) on osteoarthritic chondrocytes.

<p>(<b>A</b>) Effect of treatment of osteoarthritic chondrocytes with 25 µM cycloRGDFV peptide (RGD), control peptide RGE and additional control (untreated cells) for 24 h on SREBP-2, HMGCR and phospho-PI3K protein expression evaluated by Western blot analysis. GAPDH was utilized as loading control. (<b>B</b>) SREBP-2, HMGCR and MMP-13, and (<b>C</b>) COL2A1 and ACAN mRNA expression in osteoarthritic chondrocytes treated with RGD, RGE and respective control using Real Time PCR. GAPDH was used for normalization of the Real Time PCR data. All experiments were performed in duplicate and data are expressed as mean of two independent experiments. (<b>D</b>) MMP-13 expression levels in osteoarthritic chondrocytes following treatment with RGD, RGE and respective control detected by ELISA. *p<0.05, **p<0.001, NS: non-significant (<b>E</b>) SREBP-2 protein expression in normal and OA chondrocytes compared to genotypes (GG and GC) of the 1784G/C SNP.</p

SREBP-2 and HMGCR mRNA and protein expression in normal and osteoarthritic chondrocytes.

<p>(<b>A</b>) COL2A1 and COL1A1 ratio was screened for the verification of chondrocytes’ phenotype in cultures (<b>B</b>) SREBP-2 and HMGCR mRNA expression in normal and osteoarthritic chondrocytes. GAPDH was used for normalization of the Real Time PCR data. All experiments were performed in duplicate. *p<0.05 (<b>C</b>) Representative film showing SREBP-2 and HMGCR protein expression evaluated by Western blot analysis in normal and osteoarthritic chondrocytes. β-actin was used as loading control. (<b>D</b>) Representative film showing SREBP-2 protein expression evaluated by Western blot analysis in osteoarthritic chondrocytes treated with 25 µΜ of 25-hydroxycholesterol. GAPDH was used as loading control. (<b>E</b>) MMP-13 and COL2A1 mRNA expression in osteoarthritic chondrocytes treated with 25 µΜ of 25-hydroxycholesterol. GAPDH was used for normalization of the Real Time PCR data. All experiments were performed in duplicate. *p<0.05, NS: non significant.</p