Differential Regulation of Decorin and Biglycan Gene Expression by Dexamethasone and Retinoic Acid in Cultured Human Skin Fibroblasts

Proteoglycans participate in the assembly of extracellular matrix, directly by interacting with other matrix components and indirectly by regulating cellular growth-factor responses. We have studied the regulation of gene expression of two small extracellular matrix chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, by dexamethasone and retinoic acid In cultured human skin fibroblasts. Dexamethasone increased decorin production, maximally 4,8- fold, and decorin mRNA levels up to 2.3-fold, but had no effect on biglycan production or mRNA levels. Dexamethasone also prevented transforming growth factor-β-elicited down-regulation of decorin mRNA levels and production by dermal fibroblasts. In addition, dexamethasone potently inhibited enhancement of biglycan production and mRNA levels by transforming growth factor-β. Retinoic acid dose dependently reduced decorin mRNA levels (by 51%) and production (by 72%), but had no effect on biglycan gene expression. Retinoic acid did not alter the effect of transforming growth factor-β on decorin or biglycan production or mRNA levels. These results provide evidence that tile effects of glucocorticoids and retinoids on dermal connective tissue are partially mediated via altered expression of decorin and biglycan, which both in turn regulate the activity of transforming growth factor-β, the most potent stimulator of connective tissue deposition

Transforming Growth Factor-β Induces Collagenase-3 Expression by Human Gingival Fibroblasts via p38 Mitogen-activated Protein Kinase

Human collagenase-3 (matrix metalloproteinase 13 (MMP-13)) is characterized by exceptionally wide substrate specificity and restricted tissue specific expression. Human skin fibroblasts in culture express MMP-13 only when they are in three-dimensional collagen (Ravanti, L., Heino, J., Lopez-Otin, C., and Kahari. V.-M. (1999) J. Biol. Chem. 274, 2446-2455). Here we show that MMP-13 is expressed by fibroblasts during normal human gingival wound repair. Expression of MMP-13 by human gingival fibroblasts cultured in monolayer or in collagen gel was induced by transforming growth factor-beta1 (TGF-beta1). Treatment of gingival fibroblasts with TGF-beta1 activated two distinct mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase 1/2 (ERK1/2) in 15 min and p38 MAPK in 1 and 2 h. Induction of MMP-13 expression by TGF-beta1 was blocked by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a selective inhibitor of ERK1/2 activation. Adenovirus-mediated expression of dominant negative p38alpha and c-Jun potently inhibited induction of MMP-13 expression in gingival fibroblasts by TGF-beta1. Infection of gingival fibroblasts with adenovirus for constitutively active MEK1 resulted in activation of ERK1/2 and JNK1 and up-regulation of collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) production but did not induce MMP-13 expression. In addition, activation of p38 MAPK by constitutively active MKK6b or MKK3b was not sufficient to induce MMP-13 expression. These results show that TGF-beta-elicited induction of MMP-13 expression by gingival fibroblasts is dependent on the activity of p38 MAPK and the presence of functional AP-1 dimers. These observations demonstrate a fundamental difference in the regulation of collagenolytic capacity between gingival and dermal fibroblasts and suggest a role for MMP-13 in rapid turnover of collagenous matrix during repair of gingival wounds, which heal with minimal scarring

Contemporary radiation doses in interventional cardiology: a nationwide study of patient doses in Finland

The amount of interventional procedures such as percutaneous coronary intervention (PCI), transcatheter aortic valve implantation (TAVI), pacemaker implantation (PI) and ablations has increased within the previous decade. Simultaneously, novel fluoroscopy mainframes enable lower radiation doses for patients and operators. Therefore, there is a need to update the existing diagnostic reference levels (DRLs) and propose new ones for common or recently introduced procedures. We sought to assess patient radiation doses in interventional cardiology in a large sample from seven hospitals across Finland between 2014 and 2016. Data were used to set updated national DRLs for coronary angiographies (kerma-air product (KAP) 30 Gycm2) and PCIs (KAP 75 cm2), and novel levels for PIs (KAP 3.5 Gycm2), atrial fibrillation ablation procedures (KAP 25 Gycm2) and TAVI (KAP 90 Gycm2). Tentative KAP values were set for implantations of cardiac resynchronization therapy devices (CRT, KAP 22 Gycm2), electrophysiological treatment of atrioventricular nodal re-entry tachycardia (6 Gycm2) and atrial flutter procedures (KAP 16 Gycm2). The values for TAVI and CRT device implantation are published for the first time on national level. Dose from image acquisition (cine) constitutes the major part of the total dose in coronary and atrial fibrillation ablation procedures. For TAVI, patient weight is a good predictor of patient dose.</p

Differences in the Biosynthesis and Localization of the Fibronectin Receptor in Normal and Transformed Cultured Human Cells

We examined the biosynthesis and localization of the fibronectin receptor integrin from normal and transformed cultured human cells. Normal cells required a minimum of 20 h for the biosynthesis of completely mature fibronectin-receptor β-subunit, while transformed cells required only 6-8 h. There was a correspondingly major decrease in the amount of the intracellular β-chain precursor in the transformants. Immunostaining of normal fibroblastic cells with monoclonal antibodies indicated that both α- and β-polypeptides of the fibronectin receptor are localized in cell surface streaks and focal contact areas. In contrast, both subunits lacked this clustering and had a more diffuse distribution on the surfaces of transformed cells, even though quantitative immunofluorescence experiments indicated that similar or larger amounts of each subunit were present on a per cell basis. Both immunostaining and biochemical analyses also indicated the presence of a relatively large intracellular pool of β-polypeptides in normal fibroblasts that is not present in transformed cells. There was no major transformation-dependent change in total quantities of mature fibronectin receptor subunit expressed and inserted into the plasma membrane, when normalized to total protein synthesis. Our results indicate that malignant transformation of cultured human cells results in altered localization and processing of the fibronectin receptor. Such changes involving pathways of crucial cell surface molecules may contribute to alterations in their interactions with extracellular macromolecules, including during the process of cellular invasion. © 1990, American Association for Cancer Research. All rights reserved

Macromolecular crowding regulates matrix composition and gene expression in human gingival fibroblast cultures

Abstract Standard cell cultures are performed in aqueous media with a low macromolecule concentration compared to tissue microenvironment. In macromolecular crowding (MMC) experiments, synthetic polymeric crowders are added into cell culture media to better mimic macromolecule concentrations found in vivo. However, their effect on cultured cells is incompletely understood and appears context-dependent. Here we show using human gingival fibroblasts, a cell type associated with fast and scarless wound healing, that MMC (standard medium supplemented with Ficoll 70/400) potently modulates fibroblast phenotype and extracellular matrix (ECM) composition compared to standard culture media (nMMC) over time. MMC significantly reduced cell numbers, but increased accumulation of collagen I, cellular fibronectin, and tenascin C, while suppressing level of SPARC (Secreted Protein Acidic and Cysteine Rich). Out of the 75 wound healing and ECM related genes studied, MMC significantly modulated expression of 25 genes compared to nMMC condition. MMC also suppressed myofibroblast markers and promoted deposition of basement membrane molecules collagen IV, laminin 1, and expression of LAMB3 (Laminin Subunit Beta 3) gene. In cell-derived matrices produced by a novel decellularization protocol, the altered molecular composition of MMC matrices was replicated. Thus, MMC may improve cell culture models for research and provide novel approaches for regenerative therapy

Kindlins: essential regulators of integrin signalling and cell–matrix adhesion

Integrin-mediated cell–ECM (extracellular matrix) adhesion is a fundamental process that controls cell behaviour. For correct cell–ECM adhesion, both the ligand-binding affinity and the spatial organization of integrins must be precisely controlled; how integrins are regulated, however, is not completely understood. Kindlins constitute a family of evolutionarily conserved cytoplasmic components of cell–ECM adhesions that bind to β-integrin cytoplasmic tails directly and cooperate with talin in integrin activation. In addition, kindlins interact with many components of cell–ECM adhesions—such as migfilin and integrin-linked kinase—to promote cytoskeletal reorganization. Loss of kindlins causes severe defects in integrin signalling, cell–ECM adhesion and cytoskeletal organization, resulting in early embryonic lethality (kindlin-2), postnatal lethality (kindlin-3) and Kindler syndrome (kindlin-1). It is therefore clear that kindlins, together with several other integrin-proximal proteins, are essential for integrin signalling and cell–ECM adhesion regulation