Hispolon promotes MDM2 downregulation through chaperone-mediated autophagy.
Amplification and overexpression of murine double minute (MDM2) has been observed in several human cancers. Some chemotherapeutic agents cause MDM2 ubiquitination and degradation in a proteasome-dependent system. In addition to the proteasome system, chaperone-mediated autophagy (CMA) is a lysosomal pathway for selective misfolded protein degradation. Molecular chaperone heat shock cognate 70 protein (Hsc70) recognizes the misfolded proteins, which are then delivered to lysosome-associated membrane protein type 2A (LAMP2A) for lysosomal degradation. Our previous study reported that hispolon was able to induce cell apoptosis and downregulate MDM2 expression. In this study, our results showed that the proteasome inhibitor, MG132, could not inhibit hispolon-induced MDM2 downregulation. In contrast, both inhibition of lysosomes with NH(4)Cl and inhibition of LAMP2A using siRNA partially attenuated hispolon-induced MDM2 downregulation. To determine whether Hsc70 recognizes MDM2 on amino acids 135-141, SMP14 antibody was used to compete with Hsc70 for interaction with MDM2. After Hsc70 knockdown, SMP14 antibody immunoprecipitated increased MDM2. We also found that hispolon induced increased association of Hsp70, Hsc70, Hsp90 and LAMP2A with MDM2. This association was inhibited in cells pretreated with geldanamycin (GA), an Hsp90 inhibitor. GA also attenuated hispolon-induced MDM2 downregulation. Meanwhile, inhibition of Hsc70 using siRNA attenuated hispolon-induced MDM2 downregulation. Our study provides the first example of the ability of hispolon to mediate MDM2 downregulation in lysosomes through the CMA pathway.
Lu
Te-Ling
TL
School of Pharmacy, China Medical University, Taichung, Taiwan.
Huang
Guan-Jhong
GJ
Wang
Huang-Joe
HJ
Chen
Jian-Lian
JL
Hsu
Hui-Ping
HP
Lu
Te-Jung
TJ
D001343:Autophagy; D002396:Catechols / Q000494:pharmacology*; D002460:Cell Line; D015536:Down-Regulation; D055785:Gene Knockdown Techniques; D050883:HSC70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics
D002396:Catechols; D050883:HSC70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D018832:Molecular Chaperones; D034741:RNA, Small Interfering; C103442:hispolon; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D046988:Proteasome Endopeptidase Complex