KLF2 (kruppel-like factor 2 [lung]) regulates osteoclastogenesis by modulating autophagy

Macroautophagy/autophagy is involved in myeloid cellular repair, destruction, and osteoclast differentiation; conversely, KLF2 (kruppel-like factor 2 [lung]) regulates myeloid cell activation and differentiation. To investigate the specific role of KLF2 in autophagy, osteoclastic differentiation was induced in monocytes in presence or absence of the autophagy inhibitor 3-methyladenine (3-MA), KLF2 inducer geranylgeranyl transferase inhibitor (GGTI298), and adenoviral overexpression of KLF2. We found that the number of autophagic cells and multinucleated osteoclasts were significantly decreased in presence of 3-MA, GGTI298, and KLF2 overexpressed cells indicating involvement of KLF2 in these processes. In addition, autophagy-related protein molecules were significantly decreased after induction of KLF2 during the course of osteoclastic differentiation. Furthermore, induction of arthritis in mice reduced the level of Klf2 in monocytes, and enhanced autophagy during osteoclastic differentiation. Mechanistically, knocking down of KLF2 increased the level of Beclin1 (BECN1) expression, and conversely, KLF2 over-expression reduced the level of BECN1 in monocytes. Moreover, 3-MA and GGTI298 both reduced myeloid cell proliferation concomitantly upregulating senescence-related molecules (CDKN1A/p21 and CDKN1B/p27kip1). We further confirmed epigenetic regulation of Becn1 by modulating Klf2; knocking down of Klf2 increased the levels of histone activation marks H3K9 and H4K8 acetylation in the promoter region of Becn1; and overexpression of Klf2 decreased the levels of H4K8 and H3K9 acetylation. In addition, osteoclastic differentiation also increased levels of H3K9 and H4K8 acetylation in the promoter region of Becn1. Together these findings for the first time revealed that Klf2 critically regulates Becn1-mediated autophagy process during osteoclastogenesis. Abbreviations: ACP5/TRAP: acid phosphatase 5, tartrate resistant; Ad-KLF2: adenoviral construct of KLF2; ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; BECN1: beclin 1, autophagy related; C57BL/6: inbred mouse strain C57 black 6; ChIP: chromatin immunoprecipitation; CSF1/MCSF: colony stimulating factor 1 (macrophage); CTSK: cathepsin K; EV: empty vector; GGTI298: geranylgeranyl transferase inhibitor; H3K9Ac: histone H3 lysine 9 acetylation; H4K8Ac: histone H4 lysine 8 acetylation; K/BxN mice: T cell receptor (TCR) transgene KRN and the MHC class II molecule A(g7) generates K/BxN mice; KLF2: kruppel-like factor 2 (lung); 3MA: 3-methyladenine; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MDC: monodansylcadaverine; NFATc1: nuclear factor of activated T cells 1; NFKB: nuclear factor of kappa light polypeptide gene enhancer in B cells; p21/CDKN1A: cyclin dependent kinase inhibitor 1A; p27kip1/CDKN1B: cyclin-dependent kinase inhibitor 1B; PCR: polymerase chain reaction; PtdIns3K: phosphoinositide 3-kinase; RA: rheumatoid arthritis; siKlf2: small interfering KLF2 ribonucleic acid; NS: non-specific; RAW 264.7: abelson murine leukemia virus transformed macrophage cell line; TNFSF11/RANKL: tumor necrosis factor (ligand) superfamily, member 11; TSS: transcriptional start site; UCSC: University of California, Santa Cruz.巨自噬（Macroautophagy，即自噬（autophagy））参与髓系细胞的修复、降解以及破骨细胞分化；与之相反，KLF2（krüppel样因子2（肺型，kruppel-like factor 2 [lung]））可调控髓系细胞的活化与分化。为探究KLF2在自噬过程中的具体作用，本研究在单核细胞中诱导破骨细胞分化，并设置实验组与对照组：实验组分别添加自噬抑制剂3-甲基腺嘌呤（3-methyladenine, 3-MA）、KLF2诱导剂香叶基香叶基转移酶抑制剂（geranylgeranyl transferase inhibitor, GGTI298），以及采用腺病毒过表达KLF2，对照组则不添加相应处理。研究结果显示，在添加3-MA、GGTI298以及KLF2过表达的单核细胞中，自噬阳性细胞与多核破骨细胞的数量均显著降低，表明KLF2参与上述细胞过程。此外，在破骨细胞分化过程中，诱导KLF2表达后，自噬相关蛋白的水平显著下调。进一步研究发现，构建小鼠关节炎模型后，单核细胞内Klf2的表达水平降低，而破骨细胞分化过程中的自噬水平升高。机制研究表明，敲低KLF2可上调单核细胞中Beclin1（BECN1）的表达水平；反之，过表达KLF2则会降低单核细胞内BECN1的表达水平。此外，3-MA与GGTI298均可抑制髓系细胞增殖，同时上调衰老相关分子CDKN1A/p21与CDKN1B/p27kip1的表达水平。本研究进一步证实，Klf2可通过表观遗传调控Becn1的表达：敲低Klf2可升高Becn1启动子区域的组蛋白激活标记H3K9与H4K8乙酰化水平；而过表达Klf2则会降低H4K8与H3K9的乙酰化水平。此外，破骨细胞分化同样可升高Becn1启动子区域的H3K9与H4K8乙酰化水平。综上，本研究首次揭示Klf2在破骨细胞生成过程中，通过调控Becn1介导的自噬过程发挥关键作用。缩写说明：ACP5/TRAP：抗酒石酸酸性磷酸酶5（acid phosphatase 5, tartrate resistant）；Ad-KLF2：KLF2腺病毒构建载体（adenoviral construct of KLF2）；ATG3：自噬相关蛋白3（autophagy related 3）；ATG5：自噬相关蛋白5（autophagy related 5）；ATG7：自噬相关蛋白7（autophagy related 7）；ATG12：自噬相关蛋白12（autophagy related 12）；BECN1：自噬相关蛋白Beclin1（beclin 1, autophagy related）；C57BL/6：近交系小鼠C57黑6（inbred mouse strain C57 black 6）；ChIP：染色质免疫共沉淀（chromatin immunoprecipitation）；CSF1/MCSF：巨噬细胞集落刺激因子1（colony stimulating factor 1 (macrophage)）；CTSK：组织蛋白酶K（cathepsin K）；EV：空载体（empty vector）；GGTI298：香叶基香叶基转移酶抑制剂（geranylgeranyl transferase inhibitor）；H3K9Ac：组蛋白H3赖氨酸9乙酰化（histone H3 lysine 9 acetylation）；H4K8Ac：组蛋白H4赖氨酸8乙酰化（histone H4 lysine 8 acetylation）；K/BxN小鼠：由T细胞受体（T cell receptor, TCR）转基因KRN与MHC II类分子A(g7)构建得到的K/BxN小鼠（T cell receptor (TCR) transgene KRN and the MHC class II molecule A(g7) generates K/BxN mice）；KLF2：krüppel样因子2（肺型，kruppel-like factor 2 (lung)）；3MA：3-甲基腺嘌呤（3-methyladenine）；MAP1LC3B/LC3B：微管相关蛋白1轻链3β（microtubule-associated protein 1 light chain 3 beta）；MDC：单丹磺酰尸胺（monodansylcadaverine）；NFATc1：活化T细胞核因子1（nuclear factor of activated T cells 1）；NFKB：B细胞κ轻链增强子核因子（nuclear factor of kappa light polypeptide gene enhancer in B cells）；p21/CDKN1A：细胞周期蛋白依赖性激酶抑制剂1A（cyclin dependent kinase inhibitor 1A）；p27kip1/CDKN1B：细胞周期蛋白依赖性激酶抑制剂1B（cyclin-dependent kinase inhibitor 1B）；PCR：聚合酶链式反应（polymerase chain reaction）；PtdIns3K：磷脂酰肌醇3-激酶（phosphoinositide 3-kinase）；RA：类风湿关节炎（rheumatoid arthritis）；siKlf2：Klf2小干扰RNA（small interfering KLF2 ribonucleic acid）；NS：非特异性（non-specific）；RAW 264.7：Abelson鼠白血病病毒转化的巨噬细胞系（abelson murine leukemia virus transformed macrophage cell line）；TNFSF11/RANKL：肿瘤坏死因子（配体）超家族成员11（tumor necrosis factor (ligand) superfamily, member 11）；TSS：转录起始位点（transcriptional start site）；UCSC：加州大学圣克鲁兹分校（University of California, Santa Cruz）。