Zinc fingers are responsible for the efficient control of KLF7 on the transcription of genes in the NF-κB signaling pathway and fatty acid β-oxidation

Abstract

Krüppel-like factors (KLFs) are a family of 18 transcriptional regulators characterized by three highly conserved C2H2 zinc fingers at their C-terminal regions. KLF7, a member of this family, plays a crucial role in cell proliferation, differentiation, and the development of the nervous system, adipogenesis, diabetes, and various cancers. Studies have shown that KLF7 aggravates metabolic disorders by impeding insulin secretion and sensitivity. The nuclear factor kappa-B (NF-κB) signaling cascade is essential for inflammatory responses, while fatty acid β-oxidation is vital for metabolism. Both are linked to insulin resistance, obesity, and cardiovascular diseases. A functional link between KLF7 and the NF-κB signaling pathway has been demonstrated. In rheumatoid arthritis, KLF7 activates NF-κB signaling pathway, leading to increased cell proliferation and the production of proinflammatory cytokines, including interleukin 6 (IL-6), IL-1β, and IL-17A. In adipose tissue, KLF7 may initiate NF-κB signaling pathway by upregulating protein kinase Cζ, causing significant IL-6 secretion. KLF7 also reduces oleate-induced lipid droplets in chicken preadipocytes, indicating its role in fatty acid metabolism. Studies in mice showed that KLF7 regulates the transcription of genes of the rate-limiting glycolytic enzyme phosphofructokinase liver type (PFKL) and the fatty acid β-oxidation enzyme acyl-CoA dehydrogenase long-chain (ACADL) in cardiomyocytes independently of peroxisome proliferator-activated receptor (PPAR) γ, thereby altering heart metabolism. Additionally, KLF7 may promote cervical cancer progression by enhancing fatty acid utilization efficiency at least via ACADL upregulation. Overall, KLF7 is crucial for the regulation of fatty acid β-oxidation. KLF7 is a ring-shaped protein with zinc fingers forming the protruding part of ring surface. The lack of the third zinc finger domain in KLF7 affected its function in chicken preadipocytes. However, the importance of zinc finger domains for the regulatory function of human KLF7 remains unclear. In this study, we engineered an overexpression vector for wild-type KLF7 (pCMV-myc-KLF7_WT) and three vectors for KLF7 mutants with different zinc finger deletions (pCMV-myc-KLF7_D1, pCMV-myc-KLF7_D2, and pCMV-myc-KLF7_D3). These vectors were constructed using primers shown in Supplementary Table S1 and cDNA from HEK293T cells. Western blot analysis in the HEK293T, Ishikawa, HeLa, and EC109 cells (Pricella, Wuhan, China) showed that, unlike cells transfected with the empty vector (EV) of pCMV-myc (Clontech, Mountain View, USA), Myc-tagged proteins appeared at expected sizes in cells transfected with either the wild-type KLF7 or any of the three mutant KLF7 overexpression plasmids after 48 h (Figure 1A and Supplementary Figure S1). The impacts of overexpressing various KLF7 isoforms on gene transcription related to the NF-κB signaling pathway and fatty acid β-oxidation were evaluated using luciferase reporter assays, real-time PCR, and western blot analysis in Ishikawa, HeLa, and EC109 cells 48 h post-transfection. Details of the luciferase reporter assay transfection protocol are provided in Supplementary Table S2, the oligonucleotide sequences for real-time PCR are shown in Supplementary Table S3, and the antibodies for western blot analysis are listed in Supplementary Table S4. Compared to EV group, wild-type KLF7 overexpression significantly boosted NF-κB pathway activity in HeLa and EC109 cells (P < 0.05, Figure 1B). Furthermore, wild-type KLF7 overexpression significantly elevated IL-6 and TNF-α expressions in Ishikawa and HeLa cells (P < 0.05, Figure 1C), confirming previous findings that KLF7 enhances inflammation via the NF-κB pathway [4,5]. Cells transfected with KLF7 overexpression plasmids lacking zinc fingers showed significant differences in NF-κB pathway activities compared to those transfected with the wild-type KLF7 overexpression plasmid (P < 0.05, Figure 1B). In HeLa and EC109 cells, the absence of zinc fingers reduced NF-κB signaling activity, with the reduction proportional to the number of zinc fingers lost (P < 0.05, Figure 1B). In Ishikawa cells, losing one or two zinc fingers increased NF-κB activity compared to the wild-type KLF7 (P < 0.05, Figure 1B). Additionally, the ability of KLF7 overexpression to increase IL-6 and TNF-α expression decreased with the loss of zinc fingers.

1. Introduction

Krüppel-like factors (KLFs) are a family of 18 transcriptional regulators characterized by three highly conserved C2H2 zinc fingers at their C-terminal regions [1]. KLF7, a member of this family, plays a crucial role in cell proliferation, differentiation, and the development of the nervous system, adipogenesis, diabetes, and various cancers [2]. Studies have shown that KLF7 aggravates metabolic disorders by impeding insulin secretion and sensitivity [3].

The nuclear factor kappa-B (NF-κB) signaling cascade is essential for inflammatory responses, while fatty acid β-oxidation is vital for metabolism. Both are linked to insulin resistance, obesity, and cardiovascular diseases. A functional link between KLF7 and the NF-κB signaling pathway has been demonstrated. In rheumatoid arthritis, KLF7 activates NF-κB signaling pathway, leading to increased cell proliferation and the production of proinflammatory cytokines, including interleukin 6 (IL-6), IL-1β, and IL-17A [4]. In adipose tissue, KLF7 may initiate NF-κB signaling pathway by upregulating protein kinase Cζ, causing significant IL-6 secretion [5]. KLF7 also reduces oleate-induced lipid droplets in chicken preadipocytes, indicating its role in fatty acid metabolism [6].

Studies in mice showed that KLF7 regulates the transcription of genes of the rate-limiting glycolytic enzyme phosphofructokinase liver type (PFKL) and the fatty acid β-oxidation enzyme acyl-CoA dehydrogenase long-chain (ACADL) in cardiomyocytes independently of peroxisome proliferator-activated receptor (PPAR) γ, thereby altering heart metabolism [7]. Additionally, KLF7 may promote cervical cancer progression by enhancing fatty acid utilization efficiency at least via ACADL upregulation [8]. Overall, KLF7 is crucial for the regulation of fatty acid β-oxidation.