Increased expression of hepatocyte nuclear factor 3β (HNF3β) in hepatocellular carcinoma is associated with repression of the human organic anion transporting polypeptide 8 (OATP8) gene

Abstract

Organic anion transporting poly~ptides (OATPs) mediate the uptake ofxenobiotics, peptides and organic anions across the basolateral (sinusoidal) hepatocyte membrane. OATP-C (SLC21A6) and OATP8 (gLC21A8) are highly expressed in human liver It is unknown, however, whether expression levels are altered in hepatocellular carcinoma (HCC). The aim of this study, therefore, was to quantify the expression of OATP-C and OATP8 in eight surgically resected HCCs and to analyze the mechanism of OATP regulation in HCC. Using real*time PCR, OATP8 expression was thund to be decreased 5-50Dld in 6 out of 8 HCCs compared to surrounding non-minor liver tissue from the same patients Expression of the liver-enriched transcription factor hepatocyte nuclear factor 313 (HNF3~) was 2-50fold higher in these 6 HCCs than in non-tumor liver tissue. In one HCC, expression of HNF3[8 was decreased compared to non-tumor liver tissue and this was associated wnh increased expression of OATP8. In cnutrast to OATP8, expression of OATP-C was not significantly different in mnmr compared to non-tumor liver tissue. Sequence analysts of the 5'-flanking sequence of the OATP-C and OATP8 genes revealed consensus binding motifs for HNF31~ in both geoe promoters. Reporter gene luciterase assays in transt?cted Huh7 cells showed 70% inhibition of OATP8 promoter activity in the presence of cotransfected HNF3[3 Transfection of deletional OATP8 promoter constructs indicated that repression was mediated by an HNF3~ motif located at nt -23 to -39 t~lative to the transcription initiation site. Binding of HNF3[3 to this motif was cc, nfirmed in electrophomtic mobility Shit~ assays (EMSA). The OATP.C gene promoter was not inhibited by HNF3[3 in reporter gene assays, even though HNF3~ ,,','as shown by EMSA to bind to a proximal motif. In conclusion, HNF3~ represses tratkscription of the OATP8 but not the OATP-C gene Increased HNF3]?, expression in HCC explains reduced expression ot OATP8. On the basis of the substrate specificities of OATP-C and OATP8, these findings could permit the development of novel diagnostic and therapeutic strategies for the managemem of HCC.