Cargando…

Effects of HSD11B1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells

Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11β-HSD1 (HSD11B1) to modulate the endogenous glucocortic...

Descripción completa

Detalles Bibliográficos
Autores principales: Kragl, Angelique, Schoon, Janosch, Tzvetkova, Ana, Wenzel, Christoph, Blaschke, Martina, Böcker, Wolfgang, Siggelkow, Heide, Tzvetkov, Mladen V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9453430/
https://www.ncbi.nlm.nih.gov/pubmed/36091434
http://dx.doi.org/10.3389/fbioe.2022.953034
_version_ 1784785138526191616
author Kragl, Angelique
Schoon, Janosch
Tzvetkova, Ana
Wenzel, Christoph
Blaschke, Martina
Böcker, Wolfgang
Siggelkow, Heide
Tzvetkov, Mladen V.
author_facet Kragl, Angelique
Schoon, Janosch
Tzvetkova, Ana
Wenzel, Christoph
Blaschke, Martina
Böcker, Wolfgang
Siggelkow, Heide
Tzvetkov, Mladen V.
author_sort Kragl, Angelique
collection PubMed
description Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11β-HSD1 (HSD11B1) to modulate the endogenous glucocorticoid conversion in SCP-1 cells — a model for human mesenchymal stem cells capable of adipogenic and osteogenic differentiation. CRISPR-Cas9 was successfully used to generate a cell line carrying a single base duplication and a 5 bp deletion in exon 5, leading to missense amino acid sequences after codon 146. These inactivating genomic alterations were validated by deep sequencing and by cloning with subsequent capillary sequencing. 11β-HSD1 protein levels were reduced by 70% in the knockout cells and cortisol production was not detectable. Targeted chromosomal integration was used to stably overexpress HSD11B1. Compared to wildtype cells, HSD11B1 overexpression resulted in a 7.9-fold increase in HSD11B1 mRNA expression, a 5-fold increase in 11β-HSD1 protein expression and 3.3-fold increase in extracellular cortisol levels under adipogenic differentiation. The generated cells were used to address the effects of 11β-HSD1 expression on adipogenic and osteogenic differentiation. Compared to the wildtype, HSD11B1 overexpression led to a 3.7-fold increase in mRNA expression of lipoprotein lipase (LPL) and 2.5-fold increase in lipid production under adipogenic differentiation. Under osteogenic differentiation, HSD11B1 knockout led to enhanced alkaline phosphatase (ALP) activity and mRNA expression, and HSD11B1 overexpression resulted in a 4.6-fold and 11.7-fold increase in mRNA expression of Dickkopf-related protein 1 (DKK1) and LPL, respectively. Here we describe a HSD11B1 loss- and gain-of-function model in SCP-1 cells at genetic, molecular and functional levels. We used these models to study the effects of endogenous cortisol production on mesenchymal stem cell differentiation and demonstrate an 11β-HSD1 dependent switch from osteogenic to adipogenic differentiation. These results might help to better understand the role of endogenous cortisol production in osteoporosis on a molecular and cellular level.
format Online
Article
Text
id pubmed-9453430
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-94534302022-09-09 Effects of HSD11B1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells Kragl, Angelique Schoon, Janosch Tzvetkova, Ana Wenzel, Christoph Blaschke, Martina Böcker, Wolfgang Siggelkow, Heide Tzvetkov, Mladen V. Front Bioeng Biotechnol Bioengineering and Biotechnology Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11β-HSD1 (HSD11B1) to modulate the endogenous glucocorticoid conversion in SCP-1 cells — a model for human mesenchymal stem cells capable of adipogenic and osteogenic differentiation. CRISPR-Cas9 was successfully used to generate a cell line carrying a single base duplication and a 5 bp deletion in exon 5, leading to missense amino acid sequences after codon 146. These inactivating genomic alterations were validated by deep sequencing and by cloning with subsequent capillary sequencing. 11β-HSD1 protein levels were reduced by 70% in the knockout cells and cortisol production was not detectable. Targeted chromosomal integration was used to stably overexpress HSD11B1. Compared to wildtype cells, HSD11B1 overexpression resulted in a 7.9-fold increase in HSD11B1 mRNA expression, a 5-fold increase in 11β-HSD1 protein expression and 3.3-fold increase in extracellular cortisol levels under adipogenic differentiation. The generated cells were used to address the effects of 11β-HSD1 expression on adipogenic and osteogenic differentiation. Compared to the wildtype, HSD11B1 overexpression led to a 3.7-fold increase in mRNA expression of lipoprotein lipase (LPL) and 2.5-fold increase in lipid production under adipogenic differentiation. Under osteogenic differentiation, HSD11B1 knockout led to enhanced alkaline phosphatase (ALP) activity and mRNA expression, and HSD11B1 overexpression resulted in a 4.6-fold and 11.7-fold increase in mRNA expression of Dickkopf-related protein 1 (DKK1) and LPL, respectively. Here we describe a HSD11B1 loss- and gain-of-function model in SCP-1 cells at genetic, molecular and functional levels. We used these models to study the effects of endogenous cortisol production on mesenchymal stem cell differentiation and demonstrate an 11β-HSD1 dependent switch from osteogenic to adipogenic differentiation. These results might help to better understand the role of endogenous cortisol production in osteoporosis on a molecular and cellular level. Frontiers Media S.A. 2022-08-25 /pmc/articles/PMC9453430/ /pubmed/36091434 http://dx.doi.org/10.3389/fbioe.2022.953034 Text en Copyright © 2022 Kragl, Schoon, Tzvetkova, Wenzel, Blaschke, Böcker, Siggelkow and Tzvetkov. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Kragl, Angelique
Schoon, Janosch
Tzvetkova, Ana
Wenzel, Christoph
Blaschke, Martina
Böcker, Wolfgang
Siggelkow, Heide
Tzvetkov, Mladen V.
Effects of HSD11B1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells
title Effects of HSD11B1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells
title_full Effects of HSD11B1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells
title_fullStr Effects of HSD11B1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells
title_full_unstemmed Effects of HSD11B1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells
title_short Effects of HSD11B1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells
title_sort effects of hsd11b1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9453430/
https://www.ncbi.nlm.nih.gov/pubmed/36091434
http://dx.doi.org/10.3389/fbioe.2022.953034
work_keys_str_mv AT kraglangelique effectsofhsd11b1knockoutandoverexpressiononlocalcortisolproductionanddifferentiationofmesenchymalstemcells
AT schoonjanosch effectsofhsd11b1knockoutandoverexpressiononlocalcortisolproductionanddifferentiationofmesenchymalstemcells
AT tzvetkovaana effectsofhsd11b1knockoutandoverexpressiononlocalcortisolproductionanddifferentiationofmesenchymalstemcells
AT wenzelchristoph effectsofhsd11b1knockoutandoverexpressiononlocalcortisolproductionanddifferentiationofmesenchymalstemcells
AT blaschkemartina effectsofhsd11b1knockoutandoverexpressiononlocalcortisolproductionanddifferentiationofmesenchymalstemcells
AT bockerwolfgang effectsofhsd11b1knockoutandoverexpressiononlocalcortisolproductionanddifferentiationofmesenchymalstemcells
AT siggelkowheide effectsofhsd11b1knockoutandoverexpressiononlocalcortisolproductionanddifferentiationofmesenchymalstemcells
AT tzvetkovmladenv effectsofhsd11b1knockoutandoverexpressiononlocalcortisolproductionanddifferentiationofmesenchymalstemcells