We previously found downregulation of low-density lipoprotein receptor class A domain-containing protein 4 (LDLRAD4), a negative regulator of transforming growth factor (TGF)-beta signaling, in glutathioneS-transferase placental form (GST-P) expressing ((+)) pre-neoplastic lesions produced by treatment with nongenotoxic hepatocarcinogens for up to 90 days in rats. Here, we investigated the relationship between LDLRAD4 downregulation and TGF beta signaling in nongenotoxic hepatocarcinogenesis. The transcripts ofTgfbandHb-egfincreased after >= 28 days of treatment. After 84 or 90 days,Snai1increased transcripts and the subpopulation of GST-P(+)foci downregulating LDLRAD4 co-expressed TGF beta 1, phosphorylated EGFR, or phosphorylated AKT2, and downregulated PTEN, showing higher incidences than those in GST-P(+)foci expressing LDLRAD4. The subpopulation of GST-P(+)foci downregulating LDLRAD4 also co-expressed caveolin-1 or TACE/ADAM17, suggesting that disruptive activation of TGF beta signaling through a loss of LDLRAD4 enhances EGFR and PTEN/AKT-dependent pathways via caveolin-1-dependent activation of TACE/ADAM17 during nongenotoxic hepatocarcinogenesis. The numbers of c-MYC(+)cells and PCNA(+)cells were higher in LDLRAD4-downregulated GST-P(+)foci than in LDLRAD4-expressing GST-P(+)foci, suggesting a preferential proliferation of pre-neoplastic cells by LDLRAD4 downregulation. Nongenotoxic hepatocarcinogens markedly downregulatedNox4after 28 days and later decreased cleaved caspase 3(+)cells in LDLRAD4-downregulated GST-P(+)foci, suggesting an attenuation of apoptosis by LDLRAD4 downregulation through activation of the EGFR pathway. At the late hepatocarcinogenesis stage in a two-stage model, LDLRAD4 downregulation was higher in adenoma and carcinoma than in pre-neoplastic cell foci, suggesting a role of LDLRAD4 downregulation in tumor development. Our results suggest that nongenotoxic hepatocarcinogens cause disruptive activation of TGF beta signaling through downregulating LDLRAD4 toward carcinogenesis in the rat liver.
