Cargando…

Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity

During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage...

Descripción completa

Detalles Bibliográficos
Autores principales: Tan Timur, Ufuk, Caron, Marjolein, van den Akker, Guus, van der Windt, Anna, Visser, Jenny, van Rhijn, Lodewijk, Weinans, Harrie, Welting, Tim, Emans, Pieter, Jahr, Holger
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412363/
https://www.ncbi.nlm.nih.gov/pubmed/30781744
http://dx.doi.org/10.3390/ijms20040795
_version_ 1783402588081750016
author Tan Timur, Ufuk
Caron, Marjolein
van den Akker, Guus
van der Windt, Anna
Visser, Jenny
van Rhijn, Lodewijk
Weinans, Harrie
Welting, Tim
Emans, Pieter
Jahr, Holger
author_facet Tan Timur, Ufuk
Caron, Marjolein
van den Akker, Guus
van der Windt, Anna
Visser, Jenny
van Rhijn, Lodewijk
Weinans, Harrie
Welting, Tim
Emans, Pieter
Jahr, Holger
author_sort Tan Timur, Ufuk
collection PubMed
description During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-β superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies.
format Online
Article
Text
id pubmed-6412363
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-64123632019-04-05 Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity Tan Timur, Ufuk Caron, Marjolein van den Akker, Guus van der Windt, Anna Visser, Jenny van Rhijn, Lodewijk Weinans, Harrie Welting, Tim Emans, Pieter Jahr, Holger Int J Mol Sci Article During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-β superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies. MDPI 2019-02-13 /pmc/articles/PMC6412363/ /pubmed/30781744 http://dx.doi.org/10.3390/ijms20040795 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Tan Timur, Ufuk
Caron, Marjolein
van den Akker, Guus
van der Windt, Anna
Visser, Jenny
van Rhijn, Lodewijk
Weinans, Harrie
Welting, Tim
Emans, Pieter
Jahr, Holger
Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity
title Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity
title_full Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity
title_fullStr Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity
title_full_unstemmed Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity
title_short Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity
title_sort increased tgf-β and bmp levels and improved chondrocyte-specific marker expression in vitro under cartilage-specific physiological osmolarity
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412363/
https://www.ncbi.nlm.nih.gov/pubmed/30781744
http://dx.doi.org/10.3390/ijms20040795
work_keys_str_mv AT tantimurufuk increasedtgfbandbmplevelsandimprovedchondrocytespecificmarkerexpressioninvitroundercartilagespecificphysiologicalosmolarity
AT caronmarjolein increasedtgfbandbmplevelsandimprovedchondrocytespecificmarkerexpressioninvitroundercartilagespecificphysiologicalosmolarity
AT vandenakkerguus increasedtgfbandbmplevelsandimprovedchondrocytespecificmarkerexpressioninvitroundercartilagespecificphysiologicalosmolarity
AT vanderwindtanna increasedtgfbandbmplevelsandimprovedchondrocytespecificmarkerexpressioninvitroundercartilagespecificphysiologicalosmolarity
AT visserjenny increasedtgfbandbmplevelsandimprovedchondrocytespecificmarkerexpressioninvitroundercartilagespecificphysiologicalosmolarity
AT vanrhijnlodewijk increasedtgfbandbmplevelsandimprovedchondrocytespecificmarkerexpressioninvitroundercartilagespecificphysiologicalosmolarity
AT weinansharrie increasedtgfbandbmplevelsandimprovedchondrocytespecificmarkerexpressioninvitroundercartilagespecificphysiologicalosmolarity
AT weltingtim increasedtgfbandbmplevelsandimprovedchondrocytespecificmarkerexpressioninvitroundercartilagespecificphysiologicalosmolarity
AT emanspieter increasedtgfbandbmplevelsandimprovedchondrocytespecificmarkerexpressioninvitroundercartilagespecificphysiologicalosmolarity
AT jahrholger increasedtgfbandbmplevelsandimprovedchondrocytespecificmarkerexpressioninvitroundercartilagespecificphysiologicalosmolarity