title: β-Adducin siRNA disruption of the spectrin-based cytoskeleton in differentiating keratinocytes prevented by calcium acting through calmodulin/epidermal growth factor receptor/cadherin pathway.
abstract: Here, we report that siRNA transfection of β-adducin significantly disrupted the spectrin-based cytoskeleton and cytoskeletal arrangements of both β-adducin and PKCδ by substantially inhibiting the expression of β-adducin, spectrin and PKCδ proteins in differentiating keratinocytes. However, extracellular Ca2+ treatment blocked the inhibitory effects of the β-adducin siRNA. Ca2+ also prevented the significant down-regulation of two differentiation markers involucrin and K1/10 and the distinct up-regulation of proliferation marker K14 in β-adducin siRNA transfected keratinocytes. In addition, β-adducin knockdown resulted in a substantial reduction of epidermal growth factor receptor (EGFR), cadherin and β-catenin and enhanced phosphorylation of EGFR on tyrosine 1173 and Ca2+ prevented these changes. Furthermore, Ca2+ blocked the inhibitory effects of β-adducin siRNA on the expression of calmodulin, phosphorylated-calmodulin (P-CaM((Tyr138))) and myristoylated alanine-rich C-kinase substrate (MARCKS) in keratinocytes. Co-immunoprecipitation studies further revealed that calmodulin, not MARCKS, strongly interacted with EGFR, cadherin and β-catenin. Our data suggest that Ca2+ plays an important role in regulating the expression and function of β-adducin to sustain normal organization of the spectrin-based cytoskeleton and the differentiation properties in keratinocytes through the calmodulin/EGFR/cadherin signaling pathway.
authors.affiliation: Centre for Kidney Disease Research-Venomics Research, The University of Queensland School of Medicine, Translational Research Institute, 37 Kent Street, Woolloongabba, Brisbane, QLD 4102, Australia.
authors.forename: Jianghong
authors.initials: J
authors.lastname: Wu
authors.affiliation: Centre for Kidney Disease Research-Venomics Research, The University of Queensland School of Medicine, Translational Research Institute, 37 Kent Street, Woolloongabba, Brisbane, QLD 4102, Australia.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Masci
authors.affiliation: Centre for Kidney Disease Research-Venomics Research, The University of Queensland School of Medicine, Translational Research Institute, 37 Kent Street, Woolloongabba, Brisbane, QLD 4102, Australia.
authors.forename: Chenfeng
authors.initials: C
authors.lastname: Chen
authors.affiliation: School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.
authors.forename: Jiezhong
authors.initials: J
authors.lastname: Chen
authors.affiliation: University of Queensland Centre for Clinical Research, The University of Queensland, Herston, Brisbane, QLD 4029, Australia.
authors.forename: Martin F
authors.initials: MF
authors.lastname: Lavin
authors.affiliation: Centre for Kidney Disease Research-Venomics Research, The University of Queensland School of Medicine, Translational Research Institute, 37 Kent Street, Woolloongabba, Brisbane, QLD 4102, Australia. Electronic address: k.zhao@uq.edu.au.
authors.forename: Kong-Nan
authors.initials: KN
authors.lastname: Zhao
mesh_terms: D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D003599:Cytoskeleton / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D007633:Keratins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011498:Protein Precursors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013049:Spectrin / Q000378:metabolism*; D014162:Transfection; D019061:src-Family Kinases / Q000378:metabolism; D000099291:Adducins
chemical_list: D015820:Cadherins; D002118:Calcium; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D066246:ErbB Receptors; D047908:Intracellular Signaling Peptides and Proteins; D007633:Keratins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019869:Phosphatidylinositol 3-Kinases; D019000:Phosphotyrosine; D011498:Protein Precursors; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013049:Spectrin; D019061:src-Family Kinases; D000099291:Adducins; C000619710:MARCKS protein, human; C000619711:Marcks protein, mouse; C032253:involucrin
keywords: Cadherin; Calcium; Calmodulin; Epithelia growth factor receptor; Keratinocyte; Spectrin-based cytoskeleton; β-Adducin siRNA