Regulation of Expression and Regulated Intramembrane Proteolysis of CREB3L1



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cAMP response element binding protein 3-like 1 (CREB3L1) is a transcription factor synthesized as a membrane-bound precursor and activated through Regulated Intramembrane Proteolysis (RIP). Previous study has shown that CREB3L1 was proteolytically cleaved upon viral infection, allowing its NH2-terminus to enter nucleus to drive genes encoding inhibitors of the cell cycles. As a result, CREB3L1 inhibited replication of cells infected by some virus including Hepatitis C virus (HCV). HCV does not replicate efficiently in wild-type human hepatoma Huh-7 cells, but it replicates robustly in certain subclones of Huh-7 cells. It is found that the expression of CREB3L1 is blocked in the cells permissive for HCV replication. However, how CREB3L1 is silenced in those cells remains unknown. Here we showed that CREB3L1 and other anti-viral genes like myxovirus resistant 1 (MX1) were epigenetically silenced through DNA methylation in different subclones that are permissive for HCV replication. Methylation microarray analysis suggested that Huh-7 cells existed as a mixed population of cells with distinct patterns of gene methylation. This result indicates that subclones of Huh-7 cells become highly permissive for HCV replication by having their antiviral genes epigenetically silenced through DNA hypermethylation. In addition to driving transcription of genes encoding inhibitors of cell cycle, activation of CREB3L1 also induces the expression of genes involved in assembly of collagen matrix. Transforming growth factor-β (TGF-β) is well known to induce excessive synthesis of collagen causing tissue fibrosis. We found that sustained induction of collagen synthesis by TGF-β required proteolytic activation of CREB3L1. RIP of CREB3L1 was inhibited by transmembrane 4 L6 family member 20 (TM4SF20), which retained CREB3L1 in the endoplasmic reticulum thereby separating CREB3L1 from the Golgi-resident proteases catalyzing the RIP reaction. TGF-β inhibited TM4SF20 expression through activation of extracellular signal-regulated kinases to stimulate RIP of CREB3L1. This cleavage allowed the NH2-terminal fragment of CREB3L1 to enter the nucleus where it forms a complex with Smad4 to activate transcription of genes involved in assembly of collagen extracellular matrix. Our study suggests that RIP of CREB3L1 could be a drug target to treat tissue fibrosis. Doxorubicin, a drug used in chemotherapy, is found to inhibit cell proliferation by inducing synthesis of ceramide, which in turn activates RIP of CREB3L1. Yet the mechanism through which ceramide activates CREB3L1 is not clear. Here we reported that ceramide induced alternative translocation of TM4SF20: In the absence of ceramide, the NH2-terminus of TM4SF20 was used as signal sequence to insert the polytopic membrane protein into ER. Upon treatment of ceramide, the NH2-terminus of TM4SF20 was no longer recognized as signal sequence. Instead, the NH2-terminus of newly synthesized TM4SF20 proteins was located in the cytosol, resulting in alternative translocation of the protein that adopted a membrane topology opposite to that in the absence of the lipid. We further demonstrated that insertion of the NH2-terminal sequence of TM4SF20 into ER in the absence of ceramide required translocation associated membrane protein 2 (TRAM2), as knockdown of TRAM2 led to alternative translocation of TM4SF20 even in the absence of ceramide. Since TRAM2 contains a domain known to bind ceramide, we suspect that ceramide may inhibit the function of TRAM2. These results suggest that ceramide could induce RIP of CREB3L1 by inactivating TM4SF20 through alternative translocation.

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