Colocalized alterations in connexin32 and cytochrome P450IIB1/2 by phenobarbital and related liver tumor promoters.
Direct intercellular signal transduction is achieved by the passage of small molecules through gap junctions (GJ). Previous studies in our laboratory showed that the liver tumor promoter phenobarbital (PB) reversibly decreases the abundance of the GJ protein connexin32 (Cx32) in both preneoplastic-altered hepatic foci and centrolobular hepatocytes (M. J. Neveu et al., Cancer Commun., 2: 21-31, 1990). Because the inhibitory effects of PB on GJ intercellular communication are prevented by the nonspecific cytochrome P-450 inhibitor SKF-525A (J. E. Klauning, et al., Toxicol. Appl. Pharmacol., 102: 533-563, 1990), we investigated whether alterations in Cx32 are coincident with changes in the major PB-inducible cytochrome P-450, termed b/e or IIB1/2. Immunostaining of liver cryosections from rats fed dietary PB demonstrated that centrolobular hepatocytes that exhibit reduced Cx32 express enhanced cytochrome P450IIB1/2 protein. In contrast, no change in the periportal distribution of connexin26 immunoreactivity was found in PB-treated rats. In addition, rats were treated with the structurally related barbiturates pentobarbital, amobarbital, barbital, and barbituric acid. We found that the extent of the hepatic lobule occupied by coincident centrolobular alterations in Cx32 and P-450 staining correlates with the ability of the compounds to promote liver oncogenesis. To determine the molecular mechanisms responsible for the modification in Cx32 staining, we examined the mRNA and protein levels of Cx32 and P450IIB1/2 in total-tissue homogenates from PB-treated rats. Northern blotting demonstrated thatdietary PB dramatically induced P-450IIB1 mRNA, but the same RNA samples failed to show alterations in Cx32 steady-state transcripts. Consistent with these findings, the level of Cx32 protein in total liver homogenates did not change in rats chronically fed PB. Examination of Cx32 solubility in 20 mM NaOH demonstrated that PB treatment results in the generation of a NaOH-soluble form of Cx32 (i.e., 47 kDa). In addition, trypsinized paraffin-embedded liver sections from PB-treated rats exhibited diffuse cytoplasmic Cx32 staining that was restricted to centrolobular cells. Our results show that PB and related barbiturate tumor promoters reversibly down-regulate punctate Cx32 staining in centrolobular hepatocytes posttranslationally, possibly through modification(s) in the transport, assembly, and/or turnover of GJs.
Neveu
M J
MJ
McArdle Laboratory for Cancer Research, University of Wisconsin 53706-1599.
Babcock
K L
KL
Hertzberg
E L
EL
Paul
D L
DL
Nicholson
B J
BJ
Pitot
H C
HC
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001463:Barbiturates / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology*; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D017630:Connexins / Q000378:metabolism* / Q000502:physiology; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism* / Q000502:physiology; D004790:Enzyme Induction; D017629:Gap Junctions / Q000187:drug effects / Q000502:physiology; D015151:Immunoblotting; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000139:chemically induced / Q000201:enzymology / Q000378:metabolism; D008297:Male; D010634:Phenobarbital / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000378:metabolism* / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000097002:Gap Junction beta-1 Protein
D001189:Aryl Hydrocarbon Hydroxylases; D001463:Barbiturates; D002273:Carcinogens; D017630:Connexins; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital; D012333:RNA, Messenger; D013250:Steroid Hydroxylases; D013755:Tetradecanoylphorbol Acetate; D000097002:Gap Junction beta-1 Protein; C025619:steroid 16-beta-hydroxylase