A specific, non-immune system-related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell types
Abstract Background NOS2 expression is mostly found in bacteria-exposed or cytokine-treated tissues and is mostly connected to innate immune reactions. There are three isoforms of NOS2 (NOS2-1 to -3). In RNA-seq data sets, analyzing inflammatory gene expression, only expression of the NOS2-1 mRNA is...
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| Format: | Article |
| Language: | English |
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BMC
2022-04-01
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| Series: | Cell Communication and Signaling |
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| Online Access: | https://doi.org/10.1186/s12964-022-00855-x |
| _version_ | 1811260824897978368 |
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| author | Fabian Gather Irmgard Ihrig-Biedert Paul Kohlhas Tamara Krutenko Michael Peitz Oliver Brüstle Andrea Pautz Hartmut Kleinert |
| author_facet | Fabian Gather Irmgard Ihrig-Biedert Paul Kohlhas Tamara Krutenko Michael Peitz Oliver Brüstle Andrea Pautz Hartmut Kleinert |
| author_sort | Fabian Gather |
| collection | DOAJ |
| description | Abstract Background NOS2 expression is mostly found in bacteria-exposed or cytokine-treated tissues and is mostly connected to innate immune reactions. There are three isoforms of NOS2 (NOS2-1 to -3). In RNA-seq data sets, analyzing inflammatory gene expression, only expression of the NOS2-1 mRNA isoform is detected. However, the expression of NOS2 in differentiating human pluripotent stems (hPSCs) has not been analyzed yet. Methods Public available RNA-seq databases were screened for data of hPSCs during differentiation to different target cells. An isoform specific algorithm was used to analyze NOS2 mRNA isoform expression. In addition, we differentiated four different human iPSC cell lines toward cortical neurons and analyzed NOS2 mRNA expression by qRT-PCR and 5′-RACE. The functionality of the NOS2-2 protein was analyzed by transient transfection of expression clones in human DLD1 cells and nitrate measurement in the supernatant of these cells. Results In RNA-seq databases we detected a transient expression of the NOS2 mRNA during the differentiation of hPSCs to cardiomyocytes, chondrocytes, mesenchymal stromal cells, neurons, syncytiotrophoblast cells, and trophoblasts. NOS2 mRNA isoform specific analyses showed, that the transiently expressed NOS2 mRNA in differentiating hPSC (NOS2-2; “diff-iNOS”) differ remarkably from the already described NOS2 transcript found in colon or induced islets (NOS2-1; “immuno-iNOS”). Also, analysis of the NOS2 mRNA- and protein expression during the differentiation of four different hiPSC lines towards cortical neurons showed a transient expression of the NOS2 mRNA and NOS2 protein on day 18 of the differentiation course. 5′-RACE experiments and isoform specific qRT-PCR analyses revealed that only the NOS2-2 mRNA isoform was expressed in these experiments. To analyze the functionality of the NOS2-2 protein, we transfected human DLD-1 cells with tetracycline inducible expression clones encoding the NOS2-1- or -2 coding sequence. After induction of the NOS2-1 or -2 mRNA expression by tetracycline a similar nitrate production was measured proofing the functionality of the NOS2-2 protein isoform. Conclusions Our data show that a differentiation specific NOS2 isoform (NOS2-2) is transiently expressed during differentiation of hPSC. Video Abstract |
| first_indexed | 2024-04-12T18:53:26Z |
| format | Article |
| id | doaj.art-ed098a4fb932403ba31cdd861bb334d4 |
| institution | Directory Open Access Journal |
| issn | 1478-811X |
| language | English |
| last_indexed | 2024-04-12T18:53:26Z |
| publishDate | 2022-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell Communication and Signaling |
| spelling | doaj.art-ed098a4fb932403ba31cdd861bb334d42022-12-22T03:20:24ZengBMCCell Communication and Signaling1478-811X2022-04-0120111610.1186/s12964-022-00855-xA specific, non-immune system-related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell typesFabian Gather0Irmgard Ihrig-Biedert1Paul Kohlhas2Tamara Krutenko3Michael Peitz4Oliver Brüstle5Andrea Pautz6Hartmut Kleinert7Department of Pharmacology, University Medical Center of the Johannes Gutenberg University MainzDepartment of Pharmacology, University Medical Center of the Johannes Gutenberg University MainzDepartment of Pharmacology, University Medical Center of the Johannes Gutenberg University MainzInstitute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital BonnCell Programming Core Facility, Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital BonnInstitute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital BonnDepartment of Pharmacology, University Medical Center of the Johannes Gutenberg University MainzDepartment of Pharmacology, University Medical Center of the Johannes Gutenberg University MainzAbstract Background NOS2 expression is mostly found in bacteria-exposed or cytokine-treated tissues and is mostly connected to innate immune reactions. There are three isoforms of NOS2 (NOS2-1 to -3). In RNA-seq data sets, analyzing inflammatory gene expression, only expression of the NOS2-1 mRNA isoform is detected. However, the expression of NOS2 in differentiating human pluripotent stems (hPSCs) has not been analyzed yet. Methods Public available RNA-seq databases were screened for data of hPSCs during differentiation to different target cells. An isoform specific algorithm was used to analyze NOS2 mRNA isoform expression. In addition, we differentiated four different human iPSC cell lines toward cortical neurons and analyzed NOS2 mRNA expression by qRT-PCR and 5′-RACE. The functionality of the NOS2-2 protein was analyzed by transient transfection of expression clones in human DLD1 cells and nitrate measurement in the supernatant of these cells. Results In RNA-seq databases we detected a transient expression of the NOS2 mRNA during the differentiation of hPSCs to cardiomyocytes, chondrocytes, mesenchymal stromal cells, neurons, syncytiotrophoblast cells, and trophoblasts. NOS2 mRNA isoform specific analyses showed, that the transiently expressed NOS2 mRNA in differentiating hPSC (NOS2-2; “diff-iNOS”) differ remarkably from the already described NOS2 transcript found in colon or induced islets (NOS2-1; “immuno-iNOS”). Also, analysis of the NOS2 mRNA- and protein expression during the differentiation of four different hiPSC lines towards cortical neurons showed a transient expression of the NOS2 mRNA and NOS2 protein on day 18 of the differentiation course. 5′-RACE experiments and isoform specific qRT-PCR analyses revealed that only the NOS2-2 mRNA isoform was expressed in these experiments. To analyze the functionality of the NOS2-2 protein, we transfected human DLD-1 cells with tetracycline inducible expression clones encoding the NOS2-1- or -2 coding sequence. After induction of the NOS2-1 or -2 mRNA expression by tetracycline a similar nitrate production was measured proofing the functionality of the NOS2-2 protein isoform. Conclusions Our data show that a differentiation specific NOS2 isoform (NOS2-2) is transiently expressed during differentiation of hPSC. Video Abstracthttps://doi.org/10.1186/s12964-022-00855-xiNOSNOS2hPSCs |
| spellingShingle | Fabian Gather Irmgard Ihrig-Biedert Paul Kohlhas Tamara Krutenko Michael Peitz Oliver Brüstle Andrea Pautz Hartmut Kleinert A specific, non-immune system-related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell types Cell Communication and Signaling iNOS NOS2 hPSCs |
| title | A specific, non-immune system-related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell types |
| title_full | A specific, non-immune system-related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell types |
| title_fullStr | A specific, non-immune system-related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell types |
| title_full_unstemmed | A specific, non-immune system-related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell types |
| title_short | A specific, non-immune system-related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell types |
| title_sort | specific non immune system related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell types |
| topic | iNOS NOS2 hPSCs |
| url | https://doi.org/10.1186/s12964-022-00855-x |
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