Kruppel-like factor 15 is critical for vascular inflammation

Abstract

Activation of cells intrinsic to the vessel wall is central to the initiation and progression of vascular inflammation. As the dominant cellular constituent of the vessel wall, vascular smooth muscle cells (VSMCs) and their functions are critical determinants of vascular disease. While factors that regulate VSMC proliferation and migration have been identified, the endogenous regulators of VSMC proinflammatory activation remain incompletely defined. The Kruppel-like family of transcription factors (KLFs) are important regulators of inflammation. In this study, we identified Kruppel-like factor 15 (KLF15) as an essential regulator of VSMC proinflammatory activation. KLF15 levels were markedly reduced in human atherosclerotic tissues. Mice with systemic and smooth muscle-specific deficiency of KLF15 exhibited an aggressive inflammatory vasculopathy in two distinct models of vascular disease: orthotopic carotid artery transplantation and diet-induced atherosclerosis. We demonstrated that KLF15 alters the acetylation status and activity of the proinflammatory factor NF-κB through direct interaction with the histone acetyltransferase p300. These studies identify a previously unrecognized KLF15-dependent pathway that regulates VSMC proinflammatory activation.

Figure 1. KLF15 is decreased in human atherosclerotic tissue.

(A) KLF mRNA expression in human atherosclerotic tissue (n = 6). Values are normalized to β-actin and presented as the mean ± SEM. Insert shows individual KLF15 expression data. (B) KLF15 mRNA expression in RASMC after POVPC (100 μM) treatment for 12 hours (n = 12). Values are normalized to 18S rRNA and presented as the mean ± SD. Ao., aorta.

Figure 2. Arterial KLF15 expression reduces atherogenesis.

(A and B) Carotid arteries from WT and Klf15^–/– mice were transplanted orthotopically into congenic Apoe^–/– mice, harvested 4 weeks later, and stained with a connective tissue stain (Elastin) or a lipid stain (Oil Red O). Neointimal cross-sectional areas were measured by planimetry and plotted as the means ± SEM of greater than or equal to 6 specimens in each group. (C) Serial sections of specimens from A were immunostained for monocytes/macrophages (CD11b) or VSMCs (SMC α-actin). (D) Aortic VSMCs from WT and Klf15^–/– mice were serum starved for 24 hours and then changed into DMEM medium with no serum for 6 hours. The conditioned culture medium from different groups was subjected to ELISA for the indicated proteins. Values were normalized to VSMC protein concentration and plotted as the means ± SD. (E) Carotid grafts were harvested 1 week postoperatively, before macrophage infiltration (not shown), and frozen sections were stained for the indicated protein and DNA. The ratio of immunofluorescence/DNA fluorescence in Klf15^–/– arteries was normalized to that in WT arteries and plotted as the means ± SEM of four independent specimens of each genotype.

Figure 3. SMC-specific expression of KLF15 suppresses atherosclerosis.

(A and B) Representative image of an Smc-Klf15-KO aorta developed substantially more atherosclerotic lesions after a 15-week HFD challenge (n = 7 in the Sm22Cre/Apoe^–/– group; n = 9 in the Klf15^flox/flox/Apoe^–/– group; and n = 11 in the Smc-Klf15-KO/Apoe^–/– group). (C) Representative immunohistochemical analyses showed enhanced atherosclerotic lesion formation as well as increased MAC3, MCP-1, and VCAM-1 staining in Smc-Klf15-KO aorta. SMC α-actin staining was decreased in the Smc-Klf15-KO group (n = 7 in the Sm22Cre/Apoe^–/– group; n = 9 in the Klf15^flox/flox/Apoe^–/– group; and n = 11 in the Smc-Klf15-KO/Apoe^–/– group).

Figure 4. KLF15 suppresses NF-κB transactivation activity in VSMCs.

(A) Western blots of indicated proteins in 15-week HFD-challenged aortae nuclear extract (n = 3 individual aortae in each group). (B) Nuclear extract Western blots of AcK310-p65 and total p65 (baseline and following TNF-α stimulation) in EV and KLF15 adenovirus–infected HASMCs (left panel) and in WT and KLF15 acute knockout mouse aortic SMCs. (C) HASMCs overexpressed the indicated proteins for 48 hours, then p65 was immunoprecipitated from nuclear extracts. Immunoprecipitates were immunoblotted for total p65, total acetylated lysine (AcK), and acetylated p65-lysine310 (AcK310). (D and E) HEK293T cells were transiently transfected with the indicated plasmids for 48 hours (n = 3 independent experiments in each group). (F and G) HEK293T cells were transiently transfected by the indicated plasmids for 48 hours. p300-HAT deletion mutation showed significantly lower transcriptional activity on both MCP-1 and VCAM-1 promoters. KLF15 attenuated p300-dependent p65 activation on both promoters (n = 3 independent experiments in each group).

Figure 5. KLF15 inhibits NF-κB activation through KLF15-p300 interaction.

(A) Coimmunoprecipitation of overexpressed myc-KLF15 and endogenous p300 in HEK293T cells. (B) KLF15 overexpression prevented p300-p65 interaction in a dose-dependent fashion in HEK293T cells. (C) Immunoprecipitation of endogenous p65 and p300 after TNF-α stimulation for 20 minutes in HASMCs after acute KLF15 knockdown (left panel) and in KLF15-null MEFs (right panel). (D and E) HASMCs infected with Ad-EV or Ad-KLF15 were stimulated with TNF-α for 4 hours. ChIPs were performed with p300 antibody on MCP-1 and VCAM-1 promoters containing the NF-κB binding site. (F) The 15–amino acid KLF15 TAD domain peptide competed with full-length KLF15 for p300 immunoprecipitation in HEK293T cell nuclear extracts. (G and H) Transient transfection assays showed that the KLF15 TAD domain is critical for its repressive function. KLF15ΔTAD mutant attenuated its repressive function on MCP-1 and VCAM-1 promoters (n = three independent experiments in each group).