Osteocalcin (OC) is a bone specific protein expressed during differentiation and mineralization of the bone extracellular matrix. The Osteocalcin gene is transcriptionally regulated by several basal, hormone- and cytokine-responsive elements. To address the potential role of TGF-β1 regulation and tissue-specific expression of the OC gene, we defined regulatory elements and factors mediating the transcriptional activity of the rat OC (rOC) promoter. TGF-β1 modulates the differentiation of cells of the osteogenic lineage and downregulates the osteoblast-specific expression of OC. By promoter deletion and mutational analyses, a TGF-β1 responsive element at nt -146 to -139 and a contiguous tissue-specific enhancer element at nt -136 to -130 on the rOC promoter were identified. These studies show that Fra-2, a member of the AP-1 family of proteins, binds to the TGF-β1 responsive element and activates basal OC expression. TGF-β1 induced phosphorylation of Fra-2 inhibits this activation, resulting in repression of OC gene transcription. The tissue-specific enhancer element contiguous to the TGF-β1 responsive element contains an AML (Cbfa) binding sequence. This element, designated OC Box II, contributes to 75% of the basal OC promoter activity and forms an osteoblast-specific protein-DNA complex in in-vitro assays. The activation potential of this binding sequence was established by overexpressing AML (Cbfa) transcription activator proteins in osteoblastic as well as in non-osseous cell lines. Interestingly, overexpression not only enhances rOC promoter activity in osteoblasts but also mediates promoter activity in a non-osseous human fibroblastic cell-line. Subsequently, we identified AML-3 (Cbfa1) as the major AML family member present in the osteoblast specific complex and demonstrate that AML-3 (Cbfa1) is expressed predominantly as a 5.4 kb transcript in rat bone tissues. Finally, to establish the functional involvement of AML (Cbfa) transcription factors in osteoblast differentiation, we utilized antisense strategies to demonstrate that blocking expression of all AML (Cbfa) related genes in primary osteoblast cultures significantly decreased several parameters which are linked to differentiation of normal diploid osteoblasts. These results indicate that AML-3 (Cbfa1) is a key transcription factor in bone cells and that the activity of the AML (Cbfa) family of proteins is required for completion of osteoblast differentiation.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

High levels of lactate are positively associated with the prognosis and mortality in patients with heart attack. Endothelial-to-mesenchymal transition (EndoMT) plays an important role in cardiac fibrosis. Here, we report that lactate exerts a previously unknown function that increases cardiac fibrosis and exacerbates cardiac dysfunction by promoting EndoMT following myocardial infarction (MI). Treatment of endothelial cells with lactate disrupts endothelial cell function and induces mesenchymal-like function following hypoxia by activating the TGF-β/Smad2 pathway. Mechanistically, lactate induces an association between CBP/p300 and Snail1, leading to lactylation of Snail1, a TGF-β transcription factor, through lactate transporter monocarboxylate transporter (MCT)-dependent signaling. Inhibiting Snail1 diminishes lactate-induced EndoMT and TGF-β/Smad2 activation after hypoxia/MI. The MCT inhibitor CHC mitigates lactate-induced EndoMT and Snail1 lactylation. Silence of MCT1 compromises lactate-promoted cardiac dysfunction and EndoMT after MI. We conclude that lactate acts as an important molecule that up-regulates cardiac EndoMT after MI via induction of Snail1 lactylation.

no / This study aimed at determining the role of the transforming growth factor-beta (TGF-β) isomers and their combinations in bone cell behaviour using MG63 cells. The work examined how TGF-β1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell morphology, cell proliferation and integrin expression. This study also aimed at examining how the TGF-βs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-βs influence extracellular matrix (ECM) secretion and integrin expression. The wound healing response in terms of healing rate to the TGF-βs and their solvent/carrier was investigated in 300 μm ± 10–30 μm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The effect of different TGF-β isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. Immunostaining was used to determine if TGF-βs modifies integrin expression and ECM secretion by the bone cells. Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. The wound healing results indicated that TGF-β3 has a significant effect on the wound healing process and its healing rate was found to be higher than the control (p < 0.001), TGF-β1 (p < 0.001), TGF-β2 (p < 0.001), BSA/HCl (p < 0.001) and HCl (p < 0.001) in ascending order. It was also found that TGF-β1 and TGF-β2 treatment significantly improved wound closure rate in comparison to the controls (p < 0.001). All TGF-β combinations induced a faster healing rate than the control (p < 0.001). It was expected that the healing rate following treatment with TGF-β combinations would be greater than those healing rates following treatments with TGF-β isomers alone, but this was not the case. The results also suggest that cell morphological changes were observed significantly more in cells treated with TGF-β(2 + 3) and TGF-β(1 + 3) (p < 0.001). Any cell treated with TGF-β1, TGF-β(1 + 2) and TGF-β(1 + 2 + 3) showed significantly less elongation compared to the control and other TGF-β isomers. In terms of proliferation rate, TGF-β3 and TGF-β(2 + 3) increased cell numbers more than TGF-β1, TGF-β2 and other combinations. TGF-β1 and its combinations did not show significant proliferation and attachment compared to the control. Immunostaining indicated that treatment with TGF-β3 significantly enhanced the secretion of collagen type I, fibronectin and integrins α3 and β1. The WSPR experiments also indicated that TGF-βs influenced the distribution of focal contacts. In conclusion, combining TGF-β3 with any other TGF-β isomer resulted in a faster model wound closure rate (p < 0.001), while treatment with TGF-β1 in any TGF-β combination reduced the healing rate (p < 0.001). It can therefore be concluded that the presence of TGF-β1 has an inhibitory effect on bone wound healing while TGF-β3 had the opposite effect and increased the rate of wound closure in a 2 dimensional cell culture environment. / Emailed Mansour for final draft 27/06/2016

Osteoarthritis (OA) ist eine chronische Erkrankung der Gelenke des menschlichen Körpers, insbesondere des Kniegelenkes. Sie ist durch entzündliche und degenerative Prozesse gekennzeichnet, die Patienten in ihrer Beweglichkeit stark einschränkt. In der komplexen Pathophysiologie kommt es unter anderem zu zellmorphologischen Veränderungen der knorpelbildenden Zellen, den Chondrozyten, und zu destruktiven Veränderungen der Knorpelmatrix. Bisherige therapeutische Ansätze bestehen in meist in einer rein symptomatischen Therapie durch Schmerzmittel sowie der operativen endoprothetischen Versorgung als Ultima Ratio. Eine kurative Therapie ist bisher nicht möglich. Einen Ansatz für eine kurative Therapie könnte eine Subpopulation der Zellen des Knorpelgewebes bieten. Chondrogene Progenitor Zellen (CPCs) stellen als Vorläuferzellen der Chondrozyten, gesteuert durch das prochondrogene Gen SOX9 und das proosteogene Gen RUNX2, einen möglichen regenerativen Ansatz in der Behandlung dar. Eine Rolle in diesem Prozess könnten die Wachstumsfaktoren TGF- β3 und EGF sowie das Matrixmolekül Biglycan darstellen. In dieser Arbeit konnte gezeigt werden, dass diese Wachstumsfaktoren, deren Rezeptoren und das Matrixmolekül Biglycan im osteoarthrischen Knorpel eine Rolle spielen. Insbesondere konnte in vitro gezeigt werden, dass CPCs unter dem Einfluss dieser Moleküle zu einer vermehrten SOX9 und verminderten RUNX2-Expression angeregt werden. Unter der Hypothese, dass sich CPCs auf diese Art zu Chondrozyten differenzieren lassen und so den Knorpel wiederherstellen, könnten diese Moleküle einen möglichen Baustein einer zukünftigen Therapie der OA darstellen.