Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoter
Cancer cell migration and invasion involves temporal and spatial regulation of actin cytoskeleton reorganization, which is regulated by the WASP family of proteins such as N-WASP (Neural- Wiskott Aldrich Syndrome Protein). We have previously shown that expression of N-WASP was increased under hypoxi...
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| Format: | Article |
| Language: | English |
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Elsevier
2017-03-01
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| Series: | Biochemistry and Biophysics Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405580816302618 |
| _version_ | 1818499651770253312 |
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| author | Amrita Salvi Thirumaran Thanabalu |
| author_facet | Amrita Salvi Thirumaran Thanabalu |
| author_sort | Amrita Salvi |
| collection | DOAJ |
| description | Cancer cell migration and invasion involves temporal and spatial regulation of actin cytoskeleton reorganization, which is regulated by the WASP family of proteins such as N-WASP (Neural- Wiskott Aldrich Syndrome Protein). We have previously shown that expression of N-WASP was increased under hypoxic conditions. In order to characterize the regulation of N-WASP expression, we constructed an N-WASP promoter driven GFP reporter construct, N-WASPpro-GFP. Transfection of N-WASPpro-GFP construct and plasmid expressing HiF1α (Hypoxia Inducible factor 1α) enhanced the expression of GFP suggesting that increased expression of N-WASP under hypoxic conditions is mediated by HiF1α. Sequence analysis of the N-WASP promoter revealed the presence of two hypoxia response elements (HREs) characterized by the consensus sequence 5′-GCGTG-3′ at -132 bp(HRE1) and at -662 bp(HRE2) relative to transcription start site (TSS). Site-directed mutagenesis of HRE1(-132) but not HRE2(-662) abolished the HiF1α induced activation of N-WASP promoter. Similarly ChIP assay demonstrated that HiF1α bound to HRE1(-132) but not HRE2(-662) under hypoxic condition. MDA-MB-231 cells but not MDA-MB-231KD cells treated with hypoxia mimicking agent, DMOG showed enhanced gelatin degradation. Similarly MDA-MB-231KD(N-WASPpro-N-WASPR) cells expressing N-WASPR under the transcriptional regulation of WT N-WASPpro but not MDA-MB-231KD(N-WASPproHRE1-N-WASPR) cells expressing N-WASPR under the transcriptional regulation of N-WASPproHRE1 showed enhanced gelatin degradation when treated with DMOG. Thus indicating the importance of N-WASP in hypoxia induced invadopodia formation. Thus, our data demonstrates that hypoxia-induced activation of N-WASP expression is mediated by interaction of HiF1α with the HRE1(-132) and explains the role of N-WASP in hypoxia induced invadopodia formation. |
| first_indexed | 2024-12-10T20:32:22Z |
| format | Article |
| id | doaj.art-59dd00e3f2514581af84d95d629b28d8 |
| institution | Directory Open Access Journal |
| issn | 2405-5808 |
| language | English |
| last_indexed | 2024-12-10T20:32:22Z |
| publishDate | 2017-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biochemistry and Biophysics Reports |
| spelling | doaj.art-59dd00e3f2514581af84d95d629b28d82022-12-22T01:34:37ZengElsevierBiochemistry and Biophysics Reports2405-58082017-03-019C132110.1016/j.bbrep.2016.10.010Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoterAmrita SalviThirumaran ThanabaluCancer cell migration and invasion involves temporal and spatial regulation of actin cytoskeleton reorganization, which is regulated by the WASP family of proteins such as N-WASP (Neural- Wiskott Aldrich Syndrome Protein). We have previously shown that expression of N-WASP was increased under hypoxic conditions. In order to characterize the regulation of N-WASP expression, we constructed an N-WASP promoter driven GFP reporter construct, N-WASPpro-GFP. Transfection of N-WASPpro-GFP construct and plasmid expressing HiF1α (Hypoxia Inducible factor 1α) enhanced the expression of GFP suggesting that increased expression of N-WASP under hypoxic conditions is mediated by HiF1α. Sequence analysis of the N-WASP promoter revealed the presence of two hypoxia response elements (HREs) characterized by the consensus sequence 5′-GCGTG-3′ at -132 bp(HRE1) and at -662 bp(HRE2) relative to transcription start site (TSS). Site-directed mutagenesis of HRE1(-132) but not HRE2(-662) abolished the HiF1α induced activation of N-WASP promoter. Similarly ChIP assay demonstrated that HiF1α bound to HRE1(-132) but not HRE2(-662) under hypoxic condition. MDA-MB-231 cells but not MDA-MB-231KD cells treated with hypoxia mimicking agent, DMOG showed enhanced gelatin degradation. Similarly MDA-MB-231KD(N-WASPpro-N-WASPR) cells expressing N-WASPR under the transcriptional regulation of WT N-WASPpro but not MDA-MB-231KD(N-WASPproHRE1-N-WASPR) cells expressing N-WASPR under the transcriptional regulation of N-WASPproHRE1 showed enhanced gelatin degradation when treated with DMOG. Thus indicating the importance of N-WASP in hypoxia induced invadopodia formation. Thus, our data demonstrates that hypoxia-induced activation of N-WASP expression is mediated by interaction of HiF1α with the HRE1(-132) and explains the role of N-WASP in hypoxia induced invadopodia formation.http://www.sciencedirect.com/science/article/pii/S2405580816302618N-WASPHypoxiaHREInvadopodia |
| spellingShingle | Amrita Salvi Thirumaran Thanabalu Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoter Biochemistry and Biophysics Reports N-WASP Hypoxia HRE Invadopodia |
| title | Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoter |
| title_full | Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoter |
| title_fullStr | Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoter |
| title_full_unstemmed | Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoter |
| title_short | Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoter |
| title_sort | expression of n wasp is regulated by hif1α through the hypoxia response element in the n wasp promoter |
| topic | N-WASP Hypoxia HRE Invadopodia |
| url | http://www.sciencedirect.com/science/article/pii/S2405580816302618 |
| work_keys_str_mv | AT amritasalvi expressionofnwaspisregulatedbyhif1athroughthehypoxiaresponseelementinthenwasppromoter AT thirumaranthanabalu expressionofnwaspisregulatedbyhif1athroughthehypoxiaresponseelementinthenwasppromoter |