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

ABSTRACT

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.