Tipping the Balance of Hepatic CYP3A4 Activity Through Integrin‐Mediated Outside‐In and Inside‐Out Signaling

Abstract

Study objectiveWe have previously shown that engagement of integrin receptors by adenovirus infection or an uninfectious RGD peptide significantly suppresses hepatic cytochrome P450 3A4 (CYP3A4) and that removal of RGD epitopes from the adenovirus capsid reverses this effect. Integrin receptors are activated by two different signaling pathways. “Outside‐in” signaling is initiated when a ligand binds to the extracellular domain of the integrin receptor and “inside‐out” signaling is activated by intracellular proteins binding to the cytoplasmic domain of the β subunit of the integrin receptor. Because both pathways could be impacted by virus infection, the objective of this study is to determine how key moderators of each pathway impacts hepatic CYP3A4 expression and function.MethodsThe impact of outside‐in signaling was assessed in the human hepatocyte cell line HC‐04 through transfection of siRNA specific for the alpha and β subunits of the integrin receptor while impact of inside‐out signaling was assessed by transfection of siRNA for talin, an intracellular protein that connects the integrin β subunit to actin. CYP3A4 activity (P450‐Glo™), gene expression (qRT‐PCR) and protein levels (Western Blot) were determined after silencing was confirmed. Changes in protein levels of talin were determined by Western Blot after 24–72 hours of infection with a recombinant adenovirus (AdlacZ). Changes in gene expression pathways associated with integrins and CYP3A4 in response to silencing of integrins or talin was assessed by PCR.ResultsSilencing of the β3 subunit reversed the suppression induced by adenovirus infection. This was not observed when the alpha subunit was silenced. In contrast, silencing of talin 1 resulted in a 2.3 fold increase in CYP3A4 activity in the absence of virus infection. However, talin protein levels were reduced by 80% 48 hours after infection with AdlacZ. Changes in gene expression pathways were detected for pathways associated with integrins, talin and CYP3A4.ConclusionsOur results suggest that integrins play an important role in the regulation of hepatic CYP3A4 through the inside‐out singling pathway. Silencing of talin alone resulted in an increase in CYP3A4 activity, suggesting that the inside‐out pathway may play a role in the maintenance of baseline hepatic CYP3A4 activity and may contribute to variation in hepatic CYP3A4 activity in the human population. Understanding the interplay between integrins, talin and CYP3A4 could be used to modulate drug metabolism to improve therapeutic outcomes in healthy patients and those with disease states influenced by integrin expression such as osteoporosis, ulcerative colitis and cancer.Support or Funding InformationThis project was supported by Grant U01AI078045 (M.A.C.), a special research grant from the University of Texas Vice President for Research (M.A.C.) and a University of Texas Continuing Graduate Fellowship (K.J.‐S.).