Methyltransferase SMYD3 impairs hypoxia tolerance by augmenting hypoxia signaling independent of its enzymatic activity

Hypoxia-inducible factor (HIF)1α, a main transcriptional regulator of the cellular response to hypoxia, also plays important roles in oxygen homeostasis of aerobic organisms, which is regulated by multiple mechanisms. However, the full cellular response to hypoxia has not been elucidated. In this study, we found that expression of SMYD3, a methyltransferase, augments hypoxia signaling independent of its enzymatic activity. We demonstrated SMYD3 binds to and stabilizes HIF1α via co-immunoprecipitation and Western blot assays, leading to the enhancement of HIF1α transcriptional activity under hypoxia conditions. In addition, the stabilization of HIF1α by SMYD3 is independent of HIF1α hydroxylation by prolyl hydroxylases and the intactness of the von Hippel-Lindau ubiquitin ligase complex. Furthermore, we showed SMYD3 induces reactive oxygen species accumulation and promotes hypoxia-induced cell apoptosis. Consistent with these results, we found smyd3-null zebrafish exhibit higher hypoxia tolerance compared to their wildtype siblings. Together, these findings define a novel role of SMYD3 in affecting hypoxia signaling and demonstrate that SMYD3-mediated HIF1α stabilization augments hypoxia signaling, leading to the impairment of hypoxia tolerance. Hypoxia-inducible factor (HIF)1α, a main transcriptional regulator of the cellular response to hypoxia, also plays important roles in oxygen homeostasis of aerobic organisms, which is regulated by multiple mechanisms. However, the full cellular response to hypoxia has not been elucidated. In this study, we found that expression of SMYD3, a methyltransferase, augments hypoxia signaling independent of its enzymatic activity. We demonstrated SMYD3 binds to and stabilizes HIF1α via co-immunoprecipitation and Western blot assays, leading to the enhancement of HIF1α transcriptional activity under hypoxia conditions. In addition, the stabilization of HIF1α by SMYD3 is independent of HIF1α hydroxylation by prolyl hydroxylases and the intactness of the von Hippel-Lindau ubiquitin ligase complex. Furthermore, we showed SMYD3 induces reactive oxygen species accumulation and promotes hypoxia-induced cell apoptosis. Consistent with these results, we found smyd3-null zebrafish exhibit higher hypoxia tolerance compared to their wildtype siblings. Together, these findings define a novel role of SMYD3 in affecting hypoxia signaling and demonstrate that SMYD3-mediated HIF1α stabilization augments hypoxia signaling, leading to the impairment of hypoxia tolerance. It is well-known that oxygen profoundly affects physiology of aerobic organisms through multiple mechanisms. Molecular oxygen not only acts as the terminal electron acceptor at complex IV of the respiratory chain that yields energy during aerobic respiration and builds metabolites but also promotes to change the configuration and function of nucleic acids, sugars, lipids, proteins, and metabolites. Inadequate oxygen availability can lead to cellular dysfunction and even cell death. Under low oxygen (hypoxic) conditions, aerobic organisms utilize their cardiovascular system and respiratory system to ensure adequate oxygen delivery to cells and tissues. In addition, cells undergo adaptive changes to initiate gene expression that either enhance oxygen delivery or promote survival (1Lee P. Chandel N.S. Simon M.C. Cellular adaptation to hypoxia through hypoxia inducible factors and beyond.Nat. Rev. Mol. Cell Biol. 2020; 21: 268-283Crossref PubMed Scopus (386) Google Scholar). 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SMYD3 is a member of the SMYD lysine methylase family containing two conserved structural domains: the catalytic Su (var) 3–9, Enhancer-of-zeste, and N-terminal Trithorax (SET) domain, which is split by a Myeloid-Nervy-DEAF1 domain (33Tracy C. Warren J.S. Szulik M. Wang L. Garcia J. Makaju A. et al.The smyd family of methyltransferases: role in cardiac and skeletal muscle physiology and pathology.Curr. Opin. Physiol. 2018; 1: 140-152Crossref PubMed Scopus (56) Google Scholar). The SET domain of SMYD3 is comprised of two sections: the S-sequence, which may function as a cofactor binder as well as for protein–protein interactions, and the core SET domain, which functions as the primary catalytic location domain, and the C-terminal domain (33Tracy C. Warren J.S. Szulik M. Wang L. Garcia J. Makaju A. et al.The smyd family of methyltransferases: role in cardiac and skeletal muscle physiology and pathology.Curr. Opin. 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Novel insights into the oncogenic function of the SMYD3 lysine methyltransferase.Transl Cancer Res. 2016; 5: 330-333Crossref PubMed Scopus (6) Google Scholar). Whether or not SMYD3 involved in hypoxia signaling is still not understood. In this study, we show that SMYD3 interacts with HIF1α and stabilizes HIF1α independent of its methyltransferase activity, leading to the augment of the hypoxia signaling, the accumulation of ROS, and the enhancement of hypoxia-induced cell apoptosis. By zebrafish model, we found that disruption of smyd3 facilities zebrafish hypoxia tolerance, which might be resulted from the impact of smyd3 on hypoxia signaling. We have previously identified that the monomethyltransferase, SET7, represses hypoxia signaling by catalyzing HIF-α methylation (30Liu X. Chen Z. Xu C. Leng X. Cao H. Ouyang G. et al.Repression of hypoxia-inducible factor alpha signaling by Set7-mediated methylation.Nucl. Acids Res. 2015; 43: 5081-5098Crossref PubMed Scopus (72) Google Scholar). To determine whether other methyltransferases also involved in hypoxia signaling, initially, we examined expression of a series of methyltransferases in HEK293T cells under hypoxia. As shown in Fig. 1A, the typical hypoxia responsive genes, including GLUT1, BNIP3, PDK, PGK1, and VEGF (30Liu X. Chen Z. Xu C. Leng X. Cao H. Ouyang G. et al.Repression of hypoxia-inducible factor alpha signaling by Set7-mediated methylation.Nucl. Acids Res. 2015; 43: 5081-5098Crossref PubMed Scopus (72) Google Scholar, 31Wang J. Zhang D. Du J. Zhou C. Li Z. Liu X. et al.Tet1 facilitates hypoxia tolerance by stabilizing the HIF-alpha proteins independent of its methylcytosine dioxygenase activity.Nucl. Acids Res. 2017; 45: 12700-12714Crossref PubMed Scopus (27) Google Scholar, 45Chen Z. Liu X. Mei Z. Wang Z. Xiao W. EAF2 suppresses hypoxia-induced factor 1alpha transcriptional activity by disrupting its interaction with coactivator CBP/p300.Mol. Cell Biol. 2014; 34: 1085-1099Crossref PubMed Scopus (32) Google Scholar), were greatly induced under hypoxia, suggesting the hypoxic condition was achieved expectedly. Among the methyltransferase genes tested, SMYD2, SMYD4, SETD1A, EZH1, EZH2, and SUV420H1 were upregulated under hypoxia, but only SMYD3 was significantly suppressed (Fig. 1A), which provoked us to further test the impact of SMYD3 in affecting hypoxia signaling. Subsequently, we examined whether the effect of hypoxia on SMYD3 expression is dependent of HIF signaling. In H1299 cells, the expression of SMYD3 was significantly suppressed under hypoxia (Fig. S1A). However, in ARNT-deficient H1299 cells (ARNT−/−) (Fig. S1B), hypoxia failed to induce expression of PGK1, a typical HIF1α target gene (Fig. S1C) but could still suppress expression of SMYD3 (Fig. S1D). In addition, we added PX478 to inhibit HIF1α activity and then checked the effect of hypoxia on SMYD3 expression (46Xu C. Liu X. Zha H. Fan S. Zhang D. Li S. et al.A pathogen-derived effector modulates host glucose metabolism by arginine GlcNAcylation of HIF-1alpha protein.PLoS Pathog. 2018; 14e1007259Crossref Scopus (27) Google Scholar). When PX478 (100 μM) was added, hypoxia failed to induce expression of PGK1 (Fig. S1E) but could still suppress expression of SMYD3 (Fig. S1F). These results suggest that the effect of hypoxia on SMYD3 is independent of HIF signaling. To determine the effect of SMYD3 on hypoxia signaling, we overexpressed SMYD3 in HEK293T cells and examined expression of hypoxia responsive genes under normoxia or hypoxia. Ectopic expression of SMYD3 promoted expression of typical hypoxia responsive genes, including GLUT1, PGK1, and VEGF, under hypoxia (Fig. 1, B–D). To further confirm these observations, we changed direct-hypoxia treatment to the addition of deferoxamine mesylate salt (DFX) or CoCl2, two widely used hypoxia-mimic conditions (47Wang G.L. Semenza G.L. Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction.Blood. 1993; 82: 3610-3615Crossref PubMed Google Scholar, 48Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11715-11720Crossref PubMed Scopus (1620) Google Scholar) and then examined the effect of SMYD3 on hypoxia responsive gene expression. Consistently, overexpression of SMYD3 also enhanced expression of GLUT1, PDK1, PGK1, and BNIP3 (Fig. 1, E–J). SMYD3 is reported to downregulate the protein level of p53 (49Zhang L. Jin Y. Yang H. Li Y. Wang C. Shi Y. et al.SMYD3 promotes epithelial ovarian cancer metastasis by downregulating p53 protein stability and promoting p53 ubiquitination.Carcinogenesis. 2019; 40: 1492-1503PubMed Google Scholar), and p53 plays vital roles in hypoxia signaling (50Zhang C. Liu J. Wang J. Zhang T. Xu D. Hu W. et al.The interplay between tumor suppressor p53 and hypoxia signaling pathways in cancer.Front. Cell Dev. Biol. 2021; 9648808Google Scholar). To exclude whether the effect of SMYD3 on hypoxia signaling was mediated by p53, we examined the effect of SMYD3 on hypoxia signaling in p53-deficient H1299 cells. Similar results were obtained by H1299 cells (Fig. S1, G–I). In contrast, knockout of SMYD3 in HEK293T cell resulted in a reduction of expression of GLUT1, PGK1, PDK1, or BNIP3 under hypoxia or CoCl2 treatment (Fig. 2, A–F). Moreover, expression of Glut1 and Pgk1 was also reduced in Smyd3-deficient (Smyd3−/−) mouse embryonic fibroblast (MEF) cells compared to the wildtype MEF cells (Smyd3+/+) (Fig. 2, G–I). However, reconstitution of Smyd3 by lentivirus infection in Smyd3-/- MEF cells recovered the induction of expression of Pgk1 and Vegf compared to the empty virus control (pHAGE) (Fig. 2, J–L). HIF1α expression was confirmed by Western blot analysis (Fig. S2, A–D). In addition, knockdown of SMYD3 by shRNAs in HEK293T cell resulted in a reduction of expression of GLUT1, PDK1, or PGK1 under hypoxia (Fig. S2, E–H). Moreover, SMYD3 had similar effect on HIF2α as that on HIF1α in HEK293T cells (Fig. S3, A–F). These data suggest that SMYD3 augments hypoxia signaling. Given that HIF1α is one of the master regulators of hypoxia signaling, the enhancement of SMYD3 on hypoxia responsive gene expression promoted us to test whether this effect is mediated by HIF1α. Co-expression of SMYD3 together with HIF1α caused an induction of expression of GLUT1, PGK1, and VEGF mediated by ectopic expression of HIF1α in HEK293T cells (Fig. 3, A–C). HIF1α expression was confirmed by Western blot analysis (Fig. S4A). We next examined whether SMYD3 interacted with HIF1α. Co-immunoprecipitation assays indicated that ectopic-expressed HA-SMYD3 interacted with ectopic-expressed Myc-HIF1α (Fig. 3D). Semiendogenous co-immunoprecipitation assays indicated that ectopic-expressed HA-SMYD3 interacted with endogenous HIF1α under hypoxia (Fig. S4B). Endogenous interaction between SMYD3 and HIF1α was further confirmed in HEK293T cells under hypoxia (Fig. S4C). In Smyd3+/+ MEF cells, but not in Smyd3−/− MEF cells, endogenous Smyd3 interacted with endogenous HIF1α (Fig. 3E). Furthermore, we examined whether the protein stability of HIF1α is affected by SMYD3. Co-expression of SMYD3 together with HIF1α caused HIF1α protein level was increased steadily (Fig. 3F). Overexpression of SMYD3 upregulated endogenous HIF1α protein level under hypoxia (Fig. S4D). By contrast, the endogenous Hif1α protein level was lower in Smyd3-null MEF cells (Smyd3−/−) compared to that in Smyd3-intact MEF cells (Smyd3+/+) under hypoxia (Fig. 3G). Consistently, reconstitution of Smyd3 in Smyd3−/− MEF cells caused an increase of Hif1α protein under hypoxia (Fig. 3H). Since stabilized HIF1α needs to translocate into the nucleus to function as a transcription factor; therefore, we investigated the effect of SMYD3 on the nuclear HIF1α levels. Notably, overexpression of SMYD3 enhanced HIF1α protein in the nuclei of HEK293T cells (Fig. S4E). In agreement, Hif1α protein level was higher in the nuclei of Smyd3+/+ MEF cells compared to the nuclei of Smyd3−/− MEF cells, which was further confirmed by confocal microscopy (Fig. 3, I and J). Consistently, in cycloheximide pulse chase assay, overexpression of SMYD3 attenuated degradation of co-expressed HIF1α in HEK293T cells (Fig. S4F). These data suggest that SMYD3 interacts with and stabilizes HIF1α, leading to an increase of nuclear HIF1α and enhanced HIF1α-mediated expression of target genes. Hydroxylation of HIF1α and subsequent proteasomal degradation mediated by pVHL E3 ubiquitin ligase complex plays a central role in HIF1α regulation. We further investigated whether regulation of HIF1α by SMYD3 relies on this way. Ectopic expression of SMYD3 enhanced HIF1α protein level (Fig. S5A) and expression of GLUT1, PGK1, and PDK1 induced by addition of FG4592, a specific inhibitor of PHDs (Fig. 4, A–C) (51Rabinowitz M.H. Inhibition of hypoxia-inducible factor prolyl hydroxylase domain oxygen sensors: tricking the body into mounting orchestrated survival and repair responses.J. Med. Chem. 2013; 56: 9369-9402Crossref PubMed Scopus (127) Google Scholar). These data suggest that the induction of HIF1α target genes expression by SMYD3 might not be dependent of HIF1α hydroxylation. Furthermore, we knocked out VHL in HEK293T cells and then examined the effect of SMYD3 on hypoxia signaling (Fig. S5B). As expected, in VHL-/- HEK293T cells, the hypoxia responsive genes, including GLUT1, PGK1, PDK1, LDHA, BNIP3, PHD3, and PKM2, were increased compared to those in VHL+/+ HEK293T cells (Fig. S5C), indicating that VHL was disrupted in HEK293T cells efficiently. Ectopic expression of SMYD3 in VHL-/- HEK293T cells enhanced HIF1α protein level (Fig. S5D) and hypoxia responsive gene expression (Fig. 4, D–F) in a dose-dependent manner. These data suggest that the induction of HIF1α target genes expression by SMYD3 is independent of pVHL intactness. In addition, co-expression of SMYD3 together with HIF1α caused HIF1α protein level to increase steadily, which was not affected when the two prolyl residues (P402/P564) were mutated (HA-HIF1α-DM) (P402A/P564A) (Fig. S5, E–F). Furthermore, when FG4592 was added either in an increase of dose or in an extended time course, the protein level of endogenous Hif1α in Smyd3+/+ MEF cells kept higher than that in Smyd3-/- MEF cells (Fig. 4, G–J). Taken together, these data suggest that the induction of HIF1α target gene expression and stabilization of HIF1α by SMYD3 is independent of HIF1α hydroxylation and pVHL intactness. Given that SMYD3 serves as a methyltransferase, we sought to determine whether the modulation of HIF1α by SMYD3 was mediated by the enzymatic activity of SMYD3. Under hypoxia, ectopic expression of enzymatic-inactive mutant of SMYD3 (SMYD3-F183A) still enhanced expression of PGK1 and PDK1 in HEK293T cells, similar to its wildtype form (Fig. 5, A and B). In addition, the enzymatic-inactive mutant of SMYD3 (SMYD3-F183A) still interacted with co-expressed HIF1α under normoxia (Fig. 5C) and endogenous HIF1α under hypoxia (Fig. S6A). Consistently, overexpression of SMYD3-F183A had similar effect on co-expressed HIF1α protein stability as that of wildtype SMYD3 in either HEK293T cells or H1299 cells (Fig. 5, D and E). In addition, overexpression of SMYD3-F183A still enhanced HIF1α protein stability in H1299 cells under hypoxia (Fig. S6B). Taken together, these data suggest that SMYD3 stabilizes and activates HIF1α independent of its methyltransferase activity. Many studies have reported that reduction of the cytotoxic ROS level is associated with cell survival during hypoxia adaptation (52Kim T.H. Hur E.G. Kang S.J. Kim J.A. Thapa D. Lee Y.M. et al.NRF2 blockade suppresses colon tumor angiogenesis by inhibiting hypoxia-induced activation of HIF-1alpha.Cancer Res. 2011; 71: 2260-2275Crossref PubMed Scopus (229) Google Scholar) and that a