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Nuclear Phosphoinositide 3‐kinase delta (PIK3CD) Increases with Conditional Deletion of Hepatocellular Integrin Linked Kinase
Author(s) -
Martucci Nicole Jenna,
Cotner Mary-Clare,
Bhushan Bharat,
Mars Wendy M.,
Michalopoulos George K.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06360
Subject(s) - integrin linked kinase , chemistry , microbiology and biotechnology , hepatocyte , kinase , biology , endocrinology , protein kinase a , biochemistry , cyclin dependent kinase 2 , in vitro
The extracellular matrix (ECM) is important for survival, differentiation, and normal functioning of cells within the liver; integrins are key signaling receptors in this process. Previous data from our lab has shown that with hepatocyte specific knockout of integrin linked kinase (ILK KO), there is hepatocyte proliferation, increased matrix deposition, unorganized biliary cell/ductal proliferation, and possible transdifferentiation of hepatocytes to cholangiocytes; this led us to investigate the signaling pathways downstream of hILK KO that might be responsible for the observed phenotype, and we uncovered a potential central role for Phosphoinositide 3‐kinase (PI3K) delta (PIK3CD), a variant of PI3K that is considered to be leukocyte specific. Methods Mice with germ‐line loss of hepatocellular ILK were generated by crossing double floxed ILK mice (ILK fl/fl ) with mice expressing cre recombinase under an afp enhancer‐albumin promoter (Cre +/− ) to obtain ILK fl/fl :Cre +/− (ILK KO) and ILK fl/fl :Cre −/− (WT). Data array analyses were performed on livers from 7‐ and 14‐week old chronic ILK KO and WT mice. RNAs of interest were identified and then further examined by western blot analyses, immunohistochemical staining, and induction of acute loss of ILK using AAV8 cre in ILK fl/fl mice. Results Examination of the 7‐week RNA array analysis suggested a central role for PIK3CD in the observed hILK phenotype. Unexpectedly, using immunohistochemistry we detected prominent staining of PIK3CD protein in ILK KO hepatocytes. The presence of PIK3CD was verified in cultures of wild type hepatocytes where inhibition of PIK3CD resulted in a decrease in both pAKT (a downstream target) and Cyclin D1. Western blot analyses of whole livers also uncovered a size variant that is only observed in isolated nuclei. Additionally, at 7 days after the acute removal of ILK, we can observe staining for the cholangiocyte specific marker, CK19, in hepatocytes as well as strong PIK3CD staining in regions with spontaneous duct formation. Conclusion Our RNA and protein data from ILK KO mice have 1) revealed PIK3CD, a leukocyte specific protein, to be hepatocellular 2) shown that a size variant of PIK3CD exists in the nuclei of hepatocytes and 3) uncovered a potential relationship between PIK3CD in hepatocytes and their transdifferentiation to biliary cells. Currently, we are using an in vitro model of trans‐differentiation in an attempt to further explore these preliminary findings. Support or Funding Information CATER NIH T32 EB001026.