Expression and function of homeodomain-interacting protein kinase-2 (HIPK-2) in white and brown adipocytes

Abstract

Homeodomain-interacting protein kinase-2 (HIPK-2), a highly conserved serine/threonine nuclear kinase, has been recently identified as a central regulator of white adipocyte differentiation and white fat development. However, at present, the expression regulation and function of HIPK-2 in adipocytes remains unclear. In this study, how HIPK-2 expression is regulated, and its expression influences the differentiation of 3T3-L1 white and immortalized brown preadipocytes. Additionally, determined the lipolysis effects of HIPK-2 inhibition in differentiated 3T3-L1 cells. The effect and mode of action of known or newly synthesized HIPK-2 inhibitor on lipid accumulation and triglyceride (TG) content during the differentiation of 3T3-L1 white and brown preadipocytes were also assessed. Strikingly, HIPK-2 expression was highly induced in a time-dependent manner during the differentiation of both 3T3-L1 white and brown preadipocytes. Of interest, results of the pharmacological inhibition study demonstrated crucial roles of p38 MAPK, PKC, and PI3K/PKB in HIPK-2 overexpression during the differentiation of both 3T3-L1 white and brown preadipocytes.

Importantly, knockdown of HIPK-2 caused less lipid accumulation and TG content with no cytotoxicity during 3T3-L1 preadipocyte differentiation. Moreover, knockdown of HIPK-2 led to a decrease in not only expression of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), perilipin A, and adipokines such as adiponectin, leptin, and resistin but also phosphorylation of signal transducer and activator of transcription-3 (STAT-3) during 3T3-L1 preadipocyte differentiation. Furthermore, pharmacological inhibition of HIPK-2 by CX-4945, a known HIPK-2 inhibitor, greatly down-regulated not only lipid accumulation and triglyceride (TG) content but also C/EBP-α, PPAR-γ, FAS, acetyl-CoA carboxylase (ACC), and perilipin A during 3T3-L1 preadipocyte differentiation. CX-4945 also increased phosphorylation of cAMP-activated protein kinase (AMPK) and liver kinase-B1 (LKB-1), an upstream activator of AMPK, while decreased intracellular ATP content during 3T3-L1 preadipocyte differentiation. CX-4945 also stimulated glycerol release and phosphorylation of hormone-sensitive lipase (HSL) and extracellular signal-regulated kinase 1/2 (ERK-1/2). Of note, treatment with PD98059, an inhibitor of ERK-1/2, attenuated CX4945-induced glycerol release and HSL phosphorylation in differentiated 3T3-L1 white adipocytes, pointing out the ERK-1/2-dependent induction of lipolysis by CX-4945. CX-4945 also could greatly reduce lipid accumulation and TG content during brown preadipocyte differentiation. Strikingly, some of the novel HIPK-2 inhibitors including KMU-011401 also had the ability to strongly reduce lipid accumulation and TG content during the differentiation of both 3T3-L1 white and brown preadipocytes. These results collectively demonstrate that HIPK-2 expression is highly increased in a p38 MAPK, PKC, and PI3K/PKB-dependent manner and the increased HIPK-2 plays a crucial role in not only lipid accumulation and storage during the differentiation of both 3T3-L1 white and brown preadipocytes but also lipolysis in differentiated 3T3-L1 cells. These results advocate HIPK-2 and its inhibitor as a potential target and drug against obesity.

최근의 연구에서 homeodomain-interacting protein kinase-2 (HIPK-2)가 백색 지방세포 분화 및 지방조직 발달에 관여한다는 것이 보고되었다. 그러나 현재 백색 및 갈색 지방세포 분화 과정에서 HIPK-2의 발현 기전과 기능은 불분명하다. 이에 본 연구에서 1) 백색 및 갈색 지방전구세포의 분화 과정에서 HIPK-2 발현 조절 단백질 분석 및 HIPK-2 기능을 분석하고, 2) 분화된 백색 지방세포의 지방분해에서 HIPK-2 기능을 확인하고, 3) 기존 또는 신규 합성 HIPK-2 저해제의 백색 및 갈색 지방전구세포 분화 제어 효과 및 기전을 규명하고자 하였다. 본 연구에서 3T3-L1 백색 지방전구세포 및 갈색 지방전구세포 분화 (특히, D5 (중기) 및 D8 (말기)) 단계에서 HIPK-2 과발현이 관찰되었고, 이러한 HIPK-2 과발현이 p38 MAPK 및 PKC 활성과 깊은 관련이 있음을 확인하였다. Control shRNA transfection시킨 3T3-L1 백색 지방전구세포와 비교하여, HIPK-2 shRNA transfection시킨 세포에서 지방축적 및 중성지방 함량 감소, C/EBP-α, PPAR-γ, FAS, perilipin A 발현 감소 및 STAT-3 인산화 억제가 나타났다. 추가로 대조군 (용매 DMSO 투여시킨 3T3-L1 백색 지방전구세포)와 비교하여, 기존 HIPK-2 저해제 CX-4945 투여시킨 세포내 지방축적 및 중성지방 함량 감소, C/EBP-α, PPAR-γ, FAS, perilipin A 발현 감소, STAT-3 인산화 억제, AMPK 및 LKB-1 인산화 증가 및 ATP 함량 감소가 나타났다. 또한 분화된 3T3-L1 백색 지방세포에 CX-4945 투여 시 세포외 glycerol 방출 증가와 세포내 HSL 및 ERK-1/2 인산화가 증가되었다. 그러나 ERK-1/2 저해제 PD98059 투여 시 분화된 3T3-L1 백색 지방세포에서 CX-4945에 의한 지방분해 (glycerol 방출 및 HSL 인산화 증가)가 크게 약화되었다. 이들 결과는 CX-4945가 지방분해 효과를 가지며 이것이 ERK-1/2 의존적임을 말해주었다. 추가적으로 본 연구를 통해 신규 의약합성된 HIPK-2 저해제 KMU-011401 투여 시 분화유도제에 의한 3T3-L1 백색 지방전구세포 및 갈색 지방전구세포내 지방축적 및 중성지방 함량 증가가 크게 감소되었다. 상기 실험결과를 종합해 볼 때 백색 및 갈색 지방전구세포 분화 과정에서 일어나는 p38 MAPK 및 PKC 의존적 발현 증가된 HIPK-2는 C/EBP-α, PPAR-γ, FAS, perilipin A 발현 증가 및 STAT-3 활성화를 통해 이들 지방세포내 지방축적 및 지방저장에 중요한 기능을 수행하는 것으로 보인다. 이들 결과는 HIPK-2/HIPK-2 저해제가 향후 비만의 예방 및 치료의 잠재적 표적분자/치료제로서활용(개발)될 가능성을 제시한다.