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/p27^kip1). 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; p27^kip1/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.

Keywords: Autophagy; KLF2; histone acetylation; monocytes; osteoclast differentiation.

Figure 1.

Number of autophagic cells decreased after induction of KLF2. (a) Shows the MDC staining of RAW264.7 cells at various time points during the osteoclastic differentiation in presence or absence of 3 MA (autophagy inhibitor) or GGTI298 (KLF2 inducer), or RAW264.7 cells without differentiation as a control. (b) MDC stained vesicles were visualized in Klf2 overexpressed (Ad-Klf2) or empty vector (EV) infected control cells; and Klf2 knockdown (siKlf2) or control non-specific siRNA (NS) transfected cells during the course of osteoclast differentiation. (c) Bar graphs represent the cumulative data in percent of autophagic cells present per high power field of image. Each experiment was performed at least 3 times and 15 fields were counted/experiment. (d) Bar graphs represent the quantification of autophagic cells in (Ad-Klf2/EV) infected or (siKlf2/NS) transfected RAW264.7 cells during the course of differentiation. Each experiment was performed at least 3 times and 15 fields were counted/experiment. Data represent as Mean±SEM.

Figure 2.

Osteoclastic differentiation induced autophagic molecules and KLF2 reduced them. Western blot analysis was performed to determine changes of autophagic molecules in presence or absence of (a) 3-MA, or (b) GGTI298 or (c) adenoviral overexpression of Klf2 (Ad-Klf2) infected RAW264.7 cells during the course of osteoclastic differentiation. Undifferentiated cells were used as a control.

Figure 3.

Induction of KLF2 and reduction of autophagy inhibited osteoclastic differentiation. (a) TRAP staining was performed to determine differentiated osteoclasts from RAW264.7 cells in presence or absence of 3-MA (an autophagy inhibitor), or GGTI298 (a KLF2 inducer) during the course of osteoclastic differentiation. Undifferentiated cells were used as a control. (b) TRAP+ cells were visualized in Klf2 overexpressed (Ad-Klf2/EV) or Klf2 knockdown (NS or siKlf2) RAW264.7 cells during the course of osteoclast differentiation.(c) Bar graphs showing the cumulative data in percentage of TRAP⁺ cells present per high power field of image. Each experiment was performed at lest 3 times and 15 fields were counted/experiments. (d) Bar graphs represent the quantification of TRAP⁺ cells in Klf2 over-expressed (Ad-Klf2/EV) or Klf2 knockdown (siKlf2/NS) RAW264.7 cells during the course of differentiation. Each experiment was performed at least 3 times and 15 fields were counted/experiment. Data represent the Mean±SEM.

Figure 4.

3-MA and GGTI298 reduce cell proliferation by inducing cell senescence. (a) Cell proliferation was determined during osteoclastic differentiation after addition of 3-MA or GGTI298, and results are presented graphically. (b) Cellular senescence was detected with immunocytochemical staining with p21 and p27^kip1 after differentiation of osteoclasts (day 6) in presence of GGTI298 or 3-MA and representative images were shown.

Figure 5.

Arthritis reduces KLF2 and induces expression of osteoclast- and autophagy-related molecules. (a) Images of K/BxN serum-induced arthritic inflammation of limbs in C57/BL6 male mice. (b) Measured ankle thickness during the course of arthritis development shown graphically. Arrowheads indicate the location of measured area (ankle). (c) Expression levels of osteoclast- and autophagy-related molecules from bone marrow monocytes from RA and control mice are shown graphically after correcting with the corresponding internal control β-Actin expressions. (d) Autophagic vesicles are detected with MDC staining in differentiated osteoclasts (Day 6) from the bone marrow precursor cells of mice after induction of RA or without induction of RA as a control.

Figure 6.

KLF2 inversely regulates Beclin1 gene and protein expression. (a) Quantitative PCR analysis of Becn1 gene expression (left, upper panel) is shown graphically after over-expression of Klf2 (left lower panel) in RAW264.7 cells; and similarly expression of Becn1 gene (right, upper panel) is presented after knockdown of KLF2 (right, lower panel) in RAW264.7 cells. (b) BECN1 protein expression is depicted after overexpressing KLF2 (lower, left panel) and knocking off KLF2 (lower, right panel) in RAW264.7 cells. The levels of GAPDH were depicted as internal controls.

Figure 7.

KLF2 regulates Beclin1 gene epigenetically. Autophagy signaling pathway molecule Becn1 was analyzed using ChIP-quantitative PCR method after loss-of-function and gain-of-function of Klf2 in RAW264.7 cells. Active marks of Becn1 gene for both H3K9 acetylation (a) and H4K8 acetylation (b) were evaluated in two different promoter sites, and shown graphically. (c) H3K9 and H4K8 acetylation were also assessed after osteoclastic differentiation (on Day 6 of RAW264.7 cells), and evaluated in both promoter sites, and shown graphically. (d) KLF2 binding on Becn1 promoter regions was evaluated by KLF2-ChIP analysis in osteoclastic differentiated cells.