Leptin interferes ACTH-cAMP signaling on steroid biosynthesis in human NCI-H295 adrenocortical tumor cell line

動物體內主要分泌類固醇內泌素的腺體是性腺與腎上腺，前者分泌性類固醇，後者分泌醣皮素與鹽皮素，以維持許多生理功能的正常運作，如性類固醇調節性腺的分化與成熟，醣皮素調節醣類的代謝，鹽皮素則維持水分子及電解質的平衡等等。解剖學上，性腺與腎上腺被脂肪組織包埋著，普遍認為脂肪組織提供物理性的保護作用，然而近幾年研究發現脂肪組織所分泌的某些細胞激素，在性腺與腎上腺的生理功能上可能扮演著區域性的調節角色，其中瘦體素被證實參與性類固醇的分泌，包括對卵巢的粒性細胞、睪丸的來狄氏細胞等，瘦體素都能直接調節它們生成性類固醇。因此本研究探討瘦體素是否也參與腎上腺皮質細胞類固醇的生成，以及其可能的調控機制。 首先，利用反轉錄聚合酶鏈反應檢測出人類腎上腺皮質NCI-H295細胞能表現瘦體素接受體OB-Rb及OB-Ra。利用酵素免疫分析法發現，瘦體素處理並不影響NCI-H295細胞基底類固醇的分泌，但能顯著減少激腎上腺皮質素(ACTH)或霍亂毒素所誘發的孕酮及可體松的分泌。以西方墨點法分析類固醇生成酶P450scc、3b-HSD以及P450c21的蛋白質表現，以及以反轉錄聚合酶鏈反應分析類固醇生成基因P450scc與3b-HSD的mRNA，發現瘦體素均能減弱霍亂毒素對他們的刺激性。進一步測試P450scc啟動子活性發現，瘦體素也能減低霍亂毒素的刺激性而且作用區域在-1.7kb至-1.5kb啟動子之間。利用JAK1/2抑制劑AG490、MEK1/2抑制劑PD98059及PI3 kinase抑制劑Wortmannin測試瘦體素作用的可能訊息傳遞途徑，發現AG490及Wortmannin能防止瘦體素降低霍亂毒素的刺激作用。推測瘦體素是否能影響磷酸雙酯酶 (phosphodiesterases, PDE)活性而調節cAMP的訊息，於是利用PDE的一般性抑制劑IBMX、PDE3抑制劑SKF94836以及PDE3B無法代謝的N6-MB-cAMP測試，發現PDE3B可能是瘦體素的作用關鍵。 本論文展現瘦體素能干擾ACTH/cAMP調節腎上腺皮質細胞生成類固醇的訊息，作用層次包括孕酮與可體松的分泌、類固醇生成酶的表現、類固醇生成基因mRNA表現、以及P450scc啟動子的活性。實驗的結果顯示瘦體素可能經由JAK1/2-PI3 kinase的途徑活化PDE3B以遞解細胞內cAMP含量，因而減弱ACTH/cAMP對腎上腺皮質類固醇生成的刺激訊息。Leptin, mainly secreted from adipose tissues surrounding adrenal glands, is proposed to locally control the biosynthesis of adrenal steroid hormones. The human adrenocortical NCI-H295 cells were treated with or without leptin, adrenocorticotropic reagent, or both. Two major adrenal steroid products, progesterone and cortisol secreted in their cultured media were measured by ELISA. Cholera toxin, an activator of cAMP-protein kinase A pathway, mimicked ACTH effect to stimulate the secretion of progesterone and cortisol in time- and dose-dependent fashions. Leptin did not affect basal secretion of both steroid hormones; however, it effectively inhibited ACTH- or cholera toxin-induced secretion of progesterone and cortisol. Furthermore, the induction of cholera toxin on the protein amounts of P450scc, the first and rate-limiting steroidogenic enzyme, 3b-HSD, the essential enzyme for synthesis of bioactive steroids, and P450c21, the critical enzyme in secreting corticoids, were reduced by leptin. Similar inhibition of leptin was observed at the mRNA levels of P450scc and 3b-HSD. The involvement of leptin in regulating CYP11A1 promoter activity was evaluated by 5’-serial deletion. The deletion clones containing CYP11A1 promoter over 1.7 kb were responsive, whereas the shorter clone with 1.5-kb CYP11A1 promoter was silent to cAMP stimulation. The cAMP-inducible promoter activity was decreased by leptin. The inhibition of leptin on cAMP-regulated steroidogenesis and CYP11A1 promoter activity was prevented by the JAK1/2 specific inhibitor AG490 and PI3 kinase specific inhibitor Wortmannin as well as a general PDE inhibitor IBMX and a PDE3 selective inhibitor SKF94836; moreover, leptin failed to interfere the induction of N6-MB-cAMP, a PDE3B resistant cAMP analogue. Collectively, this study demonstrated leptin reduced adrenocorticotropic reagent-induced steroidogenesis possibly through a hypothesized JAK1/2-PI3 kinase-PDE3B-cAMP pathway.Abstract in Chinese 1 Abstract 3 Introduction 4 -Adrenal steroidogenesis 4 -Leptin 7 -Leptin receptors 8 -Leptin signaling 9 -Involvement of leptin in adrenal steroidogenesis 12 (1) Indirect effect of leptin: HPA axis 12 (2) Direct effect of leptin 12 Questions 14 Materials and Methods 15 -Cell culture and chemicals 15 -RNA extraction and cDNA synthesis 15 -Hormone assay 16 -Western blotting 16 -PCR 17 -Luciferase assay 17 -Statistical analysis 17 Results 18 -Expression of leptin receptors in NCI-H295 cells 18 -ACTH responsiveness of NCI-H295 cells 18 -Effect of leptin on basal and ACTH-regulated steroidogenesis 19 -Effect of leptin on cholera toxin-stimulated steroidogenesis 19 -Effect of leptin on protein expression of P450scc, 3b-HSD and P450c21 20 -Effect of leptin on mRNA expression of P450scc and 3b-HSD 21 -Effect of leptin on CYP11A1 promoter activity 21 -Effect of PI3 kinase inhibitor Wortmannin on leptin signaling 22 -Effect of JAK1/2 inhibitor AG490 on leptin signaling 22 -Effect of MEK1/2 inhibitor PD98059 on leptin signaling 22 -Role of PDE in leptin signaling 23 Discussions 25 References 30 Figures 41 Table 6