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Inhibition of PCSK9 Transcription by Berberine Involves Down-regulation of Hepatic HNF1α Protein Expression through the Ubiquitin-Proteasome Degradation Pathway*

Open AccessPublished:December 24, 2014DOI:https://doi.org/10.1074/jbc.M114.597229
      Our previous in vitro studies have identified hepatocyte nuclear factor 1α (HNF1α) as an obligated trans-activator for PCSK9 gene expression and demonstrated its functional involvement in the suppression of PCSK9 expression by berberine (BBR), a natural cholesterol-lowering compound. In this study, we investigated the mechanism underlying the inhibitory effect of BBR on HNF1α-mediated PCSK9 transcription. Administration of BBR to hyperlipidemic mice and hamsters lowered circulating PCSK9 concentrations and hepatic PCSK9 mRNA levels without affecting the gene expression of HNF1α. However, hepatic HNF1α protein levels were markedly reduced in BBR-treated animals as compared with the control. Using HepG2 cells as a model system, we obtained evidence that BBR treatment let to accelerated degradation of HNF1α protein. By applying inhibitors to selectively block the ubiquitin proteasome system (UPS) and autophagy-lysosomal pathway, we show that HNF1α protein content in HepG2 cells was not affected by bafilomycin A1 treatment, but it was dose-dependently increased by UPS inhibitors bortezomib and MG132. Bortezomib treatment elevated HNF1α and PCSK9 cellular levels with concomitant reductions of LDL receptor protein. Moreover, HNF1α protein displayed a multiubiquitination ladder pattern in cells treated with BBR or overexpressing ubiquitin. By expressing GFP-HNF1α fusion protein in cells, we observed that blocking UPS resulted in accumulation of GFP-HNF1α in cytoplasm. Importantly, we show that the BBR reducing effects on HNF1α protein and PCSK9 gene transcription can be eradicated by proteasome inhibitors. Altogether, our studies using BBR as a probe uncovered a new aspect of PCSK9 regulation by ubiquitin-induced proteasomal degradation of HNF1α.

      Introduction

      Mounting evidence has demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9)
      The abbreviations used are: PCSK9
      proprotein convertase subtilisin/kexin type 9
      BA1
      bafilomycin A1
      BBR
      berberine
      BTZ
      bortezomib
      HNF1α
      hepatocyte nuclear factor 1α
      LDLR
      LDL receptor
      SRE
      sterol regulatory element
      SREBP
      sterol regulatory element-binding protein
      UPS
      ubiquitin proteasome system
      LDL-C
      low density lipoprotein cholesterol
      CHX
      cycloheximide
      IP
      immunoprecipitation
      qPCR and qRT-PCR
      quantitative PCR and RT-PCR, respectively
      Ub
      ubiquitin
      DMSO
      dimethyl sulfoxide.
      is a critical player in LDL cholesterol (LDL-C) metabolism through its interaction with hepatic LDL receptor (LDLR) (
      • Peterson A.S.
      • Fong L.G.
      • Young S.G.
      PCSK9 function and physiology.
      • Qian Y.-W.
      • Schmidt R.J.
      • Zhang Y.
      • Chu S.
      • Lin A.
      • Wang H.
      • Wang X.
      • Beyer T.P.
      • Bensch W.R.
      • Bensch W.R.
      • Li W.
      • Ehsani M.E.
      • Lu D.
      • Konrad R.J.
      • Eacho P.I.
      • Moller D.E.
      • Karathanasis S.K.
      • Cao G.
      Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis.
      ,
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neutal apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
      • Seidah N.G.
      PCSK9 as a therapeutic target of dyslipidemia.
      ). PCSK9 is mainly synthesized in liver and is rapidly secreted into plasma after its maturation through a self-engaged autocatalytic cleavage in the endoplasmic reticulum (
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neutal apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
      ). PCSK9 regulates plasma LDL-C levels by diverting cell surface LDLR of hepatocytes to lysosomes for degradation (
      • Zhang D.W.
      • Lagace T.A.
      • Garuti R.
      • Zhao Z.
      • McDonald M.
      • Horton J.D.
      • Cohen J.C.
      • Hobbs H.H.
      Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation.
      ,
      • McNutt M.C.
      • Kwon H.J.
      • Chen C.
      • Chen J.R.
      • Horton J.D.
      • Lagace T.A.
      Antagonism of secreted PCSK9 increases low density lipoprotein receptor expression in HepG2 cells.
      ). Thus, PCSK9 plasma levels directly influence the level of circulating LDL-C (
      • Lambert G.
      • Ancellin N.
      • Charlton F.
      • Comas D.
      • Pilot J.
      • Keech A.
      • Patel S.
      • Sullivan D.R.
      • Cohn J.S.
      • Cohn J.S.
      • Rye K.A.
      • Barter P.J.
      Plasma PCSK9 concentration correlate with LDL and total cholesterol in diabetic patients and are decreased by fenofibrate treatment.
      ,
      • Grefhorst A.
      • McNutt M.C.
      • Lagace T.A.
      • Horton J.D.
      Plasma PCSK9 preferentially reduces liver LDL receptors in Mice.
      ). Recent successful demonstrations of neutralizing anti-PCSK9 antibodies that lowered serum LDL-C levels in dyslipidemic and hypercholesterolemic patients have provided strong validation to support the notion that lowering circulating PCSK9 levels to up-regulate hepatic LDLR is beneficial for reducing the risk of cardiovascular disease in humans (
      • Ling H.
      • Burns T.L.
      • Hilleman D.E.
      An update on the clinical development of proprotein convertase subtilisin kexin 9 inhibitors, novel therapeutic agents for lowering low-density lipoprotein cholesterol.
      ).
      In liver tissue, PCSK9 synthesis is largely controlled at the gene transcriptional level by two transcription factor families, sterol regulatory element-binding proteins (SREBPs) (
      • Horton J.D.
      • Shah N.A.
      • Warrington J.A.
      • Anderson N.N.
      • Park S.W.
      • Brown M.S.
      • Goldstein J.L.
      Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes.
      ,
      • Dubuc G.
      • Chamberland A.
      • Wassef H.
      • Davignon J.
      • Seidah N.G.
      • Bernier L.
      • Prat A.
      Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia.
      ,
      • Jeong H.J.
      • Lee H.-S.
      • Kim K.-S.
      • Kim Y.-K.
      • Yoon D.
      • Park S.W.
      Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatroy element binding protein-2.
      ) and hepatocyte nuclear factor 1 (HNF1), a dimeric transcriptional activator containing homeodomain (
      • Mendel D.B.
      • Crabtree G.R.
      HNF-1, a member of a novel class of dimerizing homeodomain proteins.
      ). PCSK9 gene expression is positively regulated by SREBP through an SRE motif of the proximal promoter in response to depletion of intracellular levels of sterols. Within the PCSK9 promoter, a highly conserved HNF1 binding site is located between the SRE and Sp1 site that functions as a tissue-specific cis-regulatory sequence of the PCSK9 promoter through the binding of the liver-enriched transcription factor HNF1α (
      • Li H.
      • Dong B.
      • Park S.W.
      • Lee H.S.
      • Chen W.
      • Liu J.
      Hepatocyte nuclear factor 1α plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine.
      ,
      • Dong B.
      • Wu M.
      • Li H.
      • Kraemer F.B.
      • Adeli K.
      • Seidah N.G.
      • Park S.W.
      • Liu J.
      Strong induction of PCSK9 gene expression through HNF1α and SREBP2: mechanism for the resistance to LDL-cholesterol lowering effect of statins in dyslipidemic hamsters.
      ,
      • Li H.
      • Liu J.
      The novel function of HINFP as a co-activator in sterol-regulated transcription of PCSK9 in HepG2 cells.
      ). We have previously reported that the interaction of HNF1α with HNF1 motif is not only requisite for the high level transcriptional activity of the PCSK9 promoter in hepatic cells; it is also a regulatory site to mediate the suppression of PCSK9 transcription by berberine (BBR), a natural cholesterol-lowering compound (
      • Kong W.
      • Wei J.
      • Abidi P.
      • Lin M.
      • Inaba S.
      • Li C.
      • Wang Y.
      • Wang Z.
      • Si S.
      • Pan H.
      • Wang S.
      • Wu J.
      • Wang Y.
      • Li Z.
      • Liu J.
      • Jiang J.D.
      Berberine is a promising novel cholesterol-lowering drug working through a unique mechanism distinct from statins.
      ). In HepG2 cells, levels of PCSK9 mRNA and protein were substantially reduced after BBR treatment (
      • Li H.
      • Dong B.
      • Park S.W.
      • Lee H.S.
      • Chen W.
      • Liu J.
      Hepatocyte nuclear factor 1α plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine.
      ,
      • Cameron J.
      • Ranheim T.
      • Kulseth M.A.
      • Leren T.P.
      • Berge K.E.
      Berberine decreases PCSK9 expression in HepG2 cells.
      ). Mutation or deletion of the HNF1 binding site on the PCSK9 promoter resulted in the loss of BBR-mediated inhibition of PCSK9 promoter activity in HepG2 cells. Likewise, siRNA-mediated depletion of intracellular HNF1α protein attenuated the suppression of PCSK9 expression by BBR treatment (
      • Li H.
      • Dong B.
      • Park S.W.
      • Lee H.S.
      • Chen W.
      • Liu J.
      Hepatocyte nuclear factor 1α plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine.
      ).
      Our subsequent in vivo study of dyslipidemic hamsters showed that BBR treatment of 100 mg/kg for 1 week lowered hepatic PCSK9 mRNA levels by 50% as compared with the PCSK9 mRNA levels in liver samples of control hamsters (
      • Dong B.
      • Wu M.
      • Li H.
      • Kraemer F.B.
      • Adeli K.
      • Seidah N.G.
      • Park S.W.
      • Liu J.
      Strong induction of PCSK9 gene expression through HNF1α and SREBP2: mechanism for the resistance to LDL-cholesterol lowering effect of statins in dyslipidemic hamsters.
      ). However, the involvement of HNF1α in BBR-mediated reduction of PCSK9 mRNA in liver tissue was not examined in that hamster study. Thus, the in vivo evidence for a functional role of HNF1α in BBR-mediated inhibition of PCSK9 gene transcription is presently lacking. Furthermore, the underlying molecular mechanisms of how BBR inhibits PCSK9 gene expression via HNF1 site remain unclear. Because inhibition of PCSK9 transcription in liver tissue will directly reduce circulating PCSK9 levels and hence lower the risk for developing cardiovascular disease, it is important to conduct further investigations to elucidate the regulatory pathway that is elicited by BBR to constrain HNF1α-mediated transactivation of PCSK9 gene expression.
      In this current study, by utilizing a hyperlipidemic mouse model, we demonstrate that BBR treatment reduced circulating PCSK9 concentrations and hepatic PCSK9 mRNA levels without affecting HNF1α gene expression. However, hepatic HNF1α protein content was greatly reduced in BBR-treated mice as compared with the control mice. Examination of liver tissues of BBR-treated hamsters further confirmed that BBR lowered HNF1α protein cellular abundance without inhibiting its gene expression. These in vivo observations from two different animal models suggest that BBR regulates HNF1α expression at translational levels. Through different lines of investigations conducted in cultured hepatic cells, we provide strong evidence to demonstrate that the ubiquitin proteasome system (UPS) is involved in BBR-mediated reduction of HNF1α protein cellular abundance, which negatively regulates PCSK9 gene transcription.

      DISCUSSION

      One important aspect of the PCSK9-LDLR pathway in mediating LDL clearance is that their transcription is coordinately regulated by sterols through a common SRE motif embedded in their gene promoters and is co-induced by current cholesterol lowering drugs, such as statins, through activation of SREBPs (
      • Horton J.D.
      • Shah N.A.
      • Warrington J.A.
      • Anderson N.N.
      • Park S.W.
      • Brown M.S.
      • Goldstein J.L.
      Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes.
      ,
      • Dubuc G.
      • Chamberland A.
      • Wassef H.
      • Davignon J.
      • Seidah N.G.
      • Bernier L.
      • Prat A.
      Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia.
      ,
      • Jeong H.J.
      • Lee H.-S.
      • Kim K.-S.
      • Kim Y.-K.
      • Yoon D.
      • Park S.W.
      Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatroy element binding protein-2.
      ). Statin treatment increases the transcription of both LDLR and PCSK9. The undesirable inducing effect of statins on PCSK9 transcription is increasingly recognized as a major limitation to statin therapeutic efficacy in further lowering plasma LDL-C (
      • Raal F.
      • Panz V.
      • Immelman A.
      • Pilcher G.
      Elevated PCSK9 levels in untreated patients with heterozygous or homozygous familial hypercholesterolemia and the response to high-dose statin therapy.
      ,
      • Costet P.
      • Hoffmann M.M.
      • Cariou B.
      • Guyomarc'h Delasalle B.
      • Konrad T.
      • Winkler K.
      Plasma PCSK9 is increased by fenofibrate and atorvastatin in a non-additive fashion in diabetic patients.
      ,
      • Welder G.
      • Zineh I.
      • Pacanowski M.A.
      • Troutt J.S.
      • Cao G.
      • Konrad R.J.
      High-dose atorvastatin causes a rapid sustained increase in human serum PCSK9 and disrupts its correlation with LDL cholesterol.
      ). Because the HNF1 binding site is unique to the PCSK9 promoter and is not present in the LDLR promoter, modulations of PCSK9 transcriptions through HNF1 sequence will not affect LDLR gene expression. Thus, the HNF1 binding site represents a divergent point to disconnect the coregulation of PCSK9 with LDLR and other SREBP target genes. Indeed, in this study, we have shown that the natural cholesterol-lowering compound BBR suppressed hepatic Pcsk9 gene expression and reduced serum PCSK9 concentrations in mice without affecting mRNA levels of LDLR and other SREBP target genes. Importantly, in the absence of an increase in gene expression, liver LDLR protein levels in BBR-treated mice were significantly higher than that of control mice, which was convoyed by a 30% reduction of serum LDL-C levels. Analysis of HNF1α mRNA and protein levels in all liver samples clearly demonstrated that BBR attenuated Pcsk9 transcription by reducing the liver content of HNF1α protein without lowering its mRNA expression. These results provided the first in vivo example of down-regulation of HNF1α alone by a lipid-lowering compound and the beneficial impact on plasma LDL-C metabolism through the PCSK9-LDLR pathway. By analyzing liver samples of control and BBR-treated hamsters, we observed essentially the same phenomenon, that BBR lowered Pcsk9 expression by reducing HNF1α protein levels. We further confirmed these in vivo findings by experiments conducted in HepG2 cells, a model human hepatic cell line. Hence, it is likely that a similar cellular mechanism is utilized by BBR to suppress PCSK9 gene expression in liver tissue in vivo and in cultured hepatic cells.
      HNF1α is a homeodomain-containing transcription factor (
      • Mendel D.B.
      • Crabtree G.R.
      HNF-1, a member of a novel class of dimerizing homeodomain proteins.
      ,
      • Cereghini S.
      Liver-enriched transcription factors and hepatocyte differentiation.
      ) that is important for diverse metabolic functions of liver, pancreatic islets, kidney, and intestine (
      • Shih D.Q.
      • Bussen M.
      • Sehayek E.
      • Ananthanarayanan M.
      • Shneider B.L.
      • Suchy F.J.
      • Shefer S.
      • Bollileni J.S.
      • Gonzalez F.J.
      • Breslow J.L.
      • Stoffel M.
      Hepatocyte nuclear factor-1α is an essential regulator of bile acid and plasma cholesterol metabolism.
      ,
      • Pontoglio M.
      • Barra J.
      • Hadchouel M.
      • Doyen A.
      • Kress C.
      • Bach J.P.
      • Babinet C.
      • Yaniv M.
      Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome.
      ,
      • Shih D.Q.
      • Screenan S.
      • Munoz K.N.
      • Philipson L.
      • Pontoglio M.
      • Yaniv M.
      • Polonsky K.S.
      • Stoffel M.
      Loss of HNF-1α function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism.
      ). Although the regulatory network that controls HNF1α gene expression has been well studied (
      • Costa R.H.
      • Kalinichenko V.V.
      • Holterman A.X.
      • Wang X.
      Transcription factors in liver development, differentiation, and regeneration.
      ,
      • Armendariz A.D.
      • Krauss R.M.
      Hepatic nuclear factor 1-α: inflammation, genetics, and atherosclerosis.
      ,
      • Pontoglio M.
      • Pausa M.
      • Doyen A.
      • Viollet B.
      • Yaniv M.
      • Tedesco F.
      Hepatocyte nuclear factor 1α controls the expression of terminal complement genes.
      ), less is known about the regulation of HNF1α at the protein level. Searching the literature, we found two reports that described the regulation of HNF1α protein. An early study conducted by Park et al. (
      • Park I.N.
      • Cho I.J.
      • Kim S.G.
      Ceramide negatively regulates glutathione S-transferase gene transactivation via repression of hepatic nuclear factor-1 that is degraded by the ubiquitin proteasome system.
      ) reported that ceramide treatment of H4IIE, a rat hepatocyte-derived cell line, repressed HNF1α protein via the ubiquitin proteasome system, whereas a recent study reported that HCV infection of human hepatoma-derived Huh-7.5 cells resulted in HNF1α protein degradation in lysosomes (
      • Matsui C.
      • Shoji I.
      • Kaneda S.
      • Sianipar I.R.
      • Deng L.
      • Hotta H.
      Hepatitis C virus infection suppresses GLUT2 gene expression via downregulation of hepatocyte nuclear factor 1α.
      ). To determine which protein degradation pathway might be operated by BBR to induce HNF1α protein degradation in HepG2 cells, initially we applied BA1 and BTZ to separately shut down the cellular protein clearance machineries. The cellular content of HNF1α was not affected by BA1 but was markedly elevated by proteasome inhibitors BTZ and MG132. It is noteworthy that the elevated HNF1α protein levels were accompanied by increased PCSK9 and reduced LDLR abundance in BTZ-treated cells. Our results provided the first evidence that the UPS-mediated degradation of HNF1α is directly linked to the PCSK9-LDLR pathway.
      BBR treatment consistently lowered HNF1α protein and PCSK9 mRNA and protein levels in HepG2 cells; however, this effect was not observed in the presence of UPS inhibitors. Both MG132 and BTZ showed antagonism to BBR-mediated repression of HNF1α protein. Utilizing BTZ, we further showed that BBR-mediated suppression of PCSK9 promoter activity was largely abolished. By co-transfection of FLAG-tagged HNF1α and Myc-tagged ubiquitin in HEK293 cells, we detected a multiubiquitination ladder pattern of HNF1α, which provided additional evidence suggesting that HNF1α is targeted by the ubiquitin-induced proteasomal degradation for its intracellular clearance. Importantly, by detection of ubiquitinated endogenous HNF1α in HepG2 cells, our study provides strong evidence showing that BBR treatment resulted in an enhanced HNF1α ubiquitination and its proteasomal degradation, which negatively impacted PCSK9 gene transcription and protein abundance in hepatic cells. Possibly, these BBR effects, at least in part, account for the LDL-C-lowering effect of BBR in hypercholesterolemic patients.
      The precise mechanism by which BBR induces HNF1α degradation via the UPS is presently unknown. Assessment of proteasome activities in HepG2 cells indicated that BBR treatment did not affect any of the three major proteolytic activities that are contained within the proteasome 20 S core. By analysis of HNF1α subcellular localization, we observed that HNF1α is accumulated in the cytoplasm upon BTZ treatment. Additionally, BTZ treatment totally reversed the reducing effect of BBR on HNF1α in the cytoplasmic fraction, but it only partially reversed the BBR effect on nuclear HNF1α. It is tempting to speculate that BBR might increase HNF1α nuclear export activity, resulting in enhanced degradation in cytoplasm. Verification of this point awaits further experimental examination.
      In summary, we have demonstrated that BBR inhibits HNF1α-mediated transactivation of PCSK9 gene expression by down-regulation of hepatic HNF1α protein content in animal models and in cultured cells where BBR treatment accelerated HNF1α protein degradation. This effect can be blocked by proteasome inhibitors that increase intracellular content of HNF1α in untreated cells. Importantly, we demonstrate that blocking the ubiquitin-proteasome pathway in HepG2 cells unfavorably affected the PCSK9-LDLR pathway by increasing PCSK9 and lowering LDLR. Considering that BTZ is currently used in the clinic as an anticancer drug (
      • Thompson J.L.
      Carfilzomib: a second-generation proteasome inhibitor for the treatment of relapsed and refractory multiple myeloma.
      ,
      • Yoshizawa K.
      • Mukai H.Y.
      • Miyazawa M.
      • Miyao M.
      • Ogawa Y.
      • Ohyashiki K.
      • Katoh T.
      • Kusumoto M.
      • Gemma A.
      • Sakai F.
      • Sugiyama Y.
      • Hatake K.
      • Fukuda Y.
      • Kudoh S.
      Bortezomib therapy-related lung disease in Japanese patients with multiple myeloma: incidence, mortality and clinical characterization.
      ) and new proteasome inhibitors are under development for treating patients with certain types of cancer, our findings in this study warrant further investigations in animal models to determine whether proteasome inhibitors would increase HNF1α protein levels in liver tissue and elevate circulating PCSK9 and LDL-C levels.

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