探討熱休克蛋白 DNAJB4/HLJ1 和熱休克因子 HSF1 調控機制在脂肪組織生成過程中所扮演之角色

Abstract

根據WHO的定義，以 BMI ≥ 30 kg/m2 為特徵的肥胖症已升級至令人擔憂的水平。預計到 2035年，全球肥胖人口將超過15億。 考慮到肥胖與非傳染性疾病(non-communicable disease)，特別是心血管疾病、各癌症、呼吸系統疾病之間的緊密聯繫，有效的肥胖管理對控制非傳染性疾病的加重影響至關重要。 由於肥胖會對脂肪細胞產生壓力，因此我們專注於了解因應這種壓力的細胞保護機制，特別是針對熱休克蛋白。 根據我們比較WT小鼠和熱休克蛋白 40 家族成員 Dnajb4 (HLJ1) 敲除小鼠 (HLJ1-/- 小鼠) 高脂飲食的初步數據，我們發現 HLJ1-/- 小鼠對飲食引起的肥胖具有增強的抵抗力。 HLJ1-/-小鼠的腹股溝白色脂肪組織 (iWAT) 中的 SCD1 蛋白和 mRNA升高。 SCD1 可以產生單元不飽和脂肪酸，而單元不飽和脂肪酸可以增強sirtuins蛋白的活性，進而增強脂肪分解。 鑑於 SCD1 promoter中存在預測的 HSE序列，顯示 SCD1 轉錄預計由 HSF1 調節。 然而，在高脂環境壓力下，脂肪細胞中HLJ1對HSF1的調節機制仍不清楚。 我們假設 HLJ1 的缺失會提高 HSF1 的活性和表達，可作為透過增強脂肪細胞分解脂肪以實現抗肥胖效果的標靶治療。 在本研究中，我們建構了 3 個主要質體，用於僅過度 HSF1表現、用HSF1和HLJ1 共同過度表現以及由 SCD1 promoter驅動的螢光素酶luciferase質體，將用於後續實驗。 透過螢光素酶Luciferase和cycloheximide測定，我們發現HLJ1分別降低HSF1轉錄活性和半衰期，顯示有負調控。 我們在 MG132 抑制下觀察到免疫沉澱樣本中 HLJ1 和 HSF1 的變化，顯示ubiquitination參與了它們的調節。 然而，HSF1和HLJ1之間的直接物理結合或詳細的調控機制仍沒有定論。 此外，我們的研究也發現 HSF1 和 HLJ1 對脂肪細胞生物學中的關鍵基因具有拮抗作用。因此，HSF1和HLJ1之間的詳細關係和機制仍繼續研究。 本研究旨在揭示HLJ1和HSF1之間作用的分子機制，特別是它們于脂肪生成的調控，為未來肥胖管理提供潛在的標靶。

Obesity, defined by a body mass index (BMI) above 30 kg/m2, has escalated to a concerning level, with over 1.53 billion people estimated to be affected in 2035, according to the world obesity federation. Its association with non-communicable diseases (NCDs), such as cardiovascular diseases, cancers, respiratory disorders, and diabetes, emphasizes the urgent need for effective management strategies. As obesity induces adipocyte stress, we focus on understanding cellular protective mechanisms in response to this stress, specifically targeting heat shock proteins (HSPs). Our preliminary data, comparing wild-type mice (WT mice) and a heat shock protein 40 family member Dnajb4 (HLJ1) knock-out mice (HLJ1-/- mice) fed with high-fat diets, revealed that HLJ1-/- mice exhibited increased resistance to diet-induced obesity. The inguinal white adipose tissues (iWATs) of HLJ1-/- mice had elevated of Stearoyl-CoA desaturase 1 (SCD1) protein and mRNA expression levels. SCD1 has been known to generate monounsaturated fatty acids, which can enhance sirtuin activity. Our findings suggested that the elevated SCD1 transcription could be regulated by heat shock transcription factor 1 (HSF1), given the predicted heat shock element (HSE) in the SCD1 promoter region. However, the regulatory mechanism of HSF1 by HLJ1 in adipocytes under high-fat environment stress remains unclear. We hypothesize that the loss of HLJ1 increases the HSF1 activity and its expression level, potentially emerging as a therapeutic target through enhancing adipocyte lipolysis to achieve an anti-obesity effect. For subsequent experiments in this study, plasmids including HSF1 overexpression, HSF1-HLJ1 co-overexpression, or luciferase reporter plasmids under SCD1 promoter (with or without predicted HSE) were constructed. Luciferase and cycloheximide assays revealed HLJ1-mediated reduction in HSF1 transcriptional activity and stability, suggesting the negative regulation. Immunoprecipitation analysis under proteasome inhibitor MG132 indicated potential involvement of post-translational modifications, possibly ubiquitination, in HLJ1 and HSF1 regulation. However, the direct physical binding between HSF1 and HLJ1 or the detailed regulatory mechanism remains inconclusive. Furthermore, our investigation revealed that HSF1 and HLJ1 exert antagonistic effects on essential genes crucial for adipocyte biology. This study uncovered the molecular mechanisms underlying the relationship between HLJ1 and HSF1, particularly their roles in adipocyte biology, to provide a potential target for obesity management in the future.