쥐 고환 간질세포에서 Cadmium이 스테로이드 합성의 조절에 미치는 영향 = (The)Effect of Cadmium on Regulation of the Steroidogenesis in Mouse Testicular Leyding Cells

다국어 초록 (Multilingual Abstract)

Cadmium is one of the widely used industrially and environmentally dispersed heavy metal that affects the male reproductive system of animals including human. However, the molecular mechanism of its effect on the steroidogenesis in gonad remains unclear. In this study, we demonstrated that cadmium exposure to K28 mouse testicular leydig tumor cells led to a significant increase in mRNA level, promoter activity and protein level of the steroidogenic acute regulatory protein (StAR), an essential factor for steroid biosynthesis in leydig cells. Cadmium-induced StAR protein level was completely blocked by the PKA and PKC signaling inhibitors. It has been well documented that StAR gene transcription is regulated by multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members and SF-1. Cadmium treatment caused increase in CREB phosphorylation but did not change nuclear CREB protein level. ChIP and EMSA studies revealed that cadmium-induced phosphorylated-CREB binds to the proximal region of StAR gene promoter. Furthermore, co-transfection with CREB expression plasmid significantly increased the cadmium-induced StAR promoter activity. However, nuclear level and affinity of SF-1 protein to the StAR proximal promoter was dramatically decreased upon exposure to cadmium. Taken together, these results suggest that cadmium up-regulates the StAR gene expression through CREB phosphorylation rather than through SF-1 in mouse testicular leydig cells. In this study, we also demonstrated that cadmium exposure to K28 mouse testicular leydig tumor cells led to a significant decrease in pregnenolone and progesterone production. Moreover, cadmium exposure significantly diminished protein expression level of mitochondrial cytochrome P450 side chain cleavage (P450scc) and phosphorylation of hormone-sensitive lipase (HSL). However, no significant changes were observed in protein expression levels of 3β-HSD, P450 17α, HSL. The scavenger receptor class B type 1 (SR-B1), an essential factor for uptake of cholesterol ester in leydig cells was dramatically inecreased upon exposure to cadmium. Taken together, these results suggest that cadmium inhibits progesterone production through suppression of P450scc expression and HSL phosphorylation in mouse testicular leydig cells.

국문 초록 (Abstract)

Cadmium 은 산업에 널리 사용되며 환경에 흩어져있는 중금속 중의 하나로 인간을 포함한 동물의 남성 생식계에 영향을 미친다. 그러나 생식선에서의 스테로이드 합성에 이것이 미치는 분자적 기작은 명백하지 않다. 본 연구에서, K28 쥐 고환 간질 세포에 cadmium 노출은 간질세포에서 스테로이드 생합성에 있어 중요한 요소인 스테로이드 급성 합성 단백질 (StA R)의 mRNA 와 단백질 수준, prompter 활성의 의미있는 증가를 이끌었다. Cadmium 에 의해 유도된 StAR 단백질은 PKA 와 PKC 억제제에 의해 완벽하게 저해되었다. StAR 유전자 전사는 CREB 집단 구성요소와 SF-1 을 포함한 다양한 전사 인자 들에 의해 조절됨은 잘 정리되어있다. Cadmium 처리는 CREB 의 인산화를 증가시키지만 핵내 CREB 단백질 정도는 변화 시키지 않았다. Cadmium 에 의해 유도된 인산화된 CREB 단백질이 StAR promoter 근위부와 상호작용함이 ChIP 과 EMSA 실험을 통해 들러났다. 게다가, Cadmium 에 의해 유도된 StAR prompter 활성이 CREB 발현 plasmid 를 같이 transfection 했을 때 더욱 증가했다. 이러한 결과들을 종합하면, Cadmium 이 StAR 를 상향 조절하는 것은 SF-1 보다 인산화된 CREB 을 통해서라고 제안할 수 있다.
또한 이 본 연구에서, K28 쥐 고환 간질 세포에 cadmium 노출은 pregnenolone 과 progesterone 생성을 크게 감소시켰다. 게다가, cadmium 노출은 미토콘드리아내의 P450scc 단백질 발현과 HSL 의 인산화를 크게 감소시켰다.
그러나 3β-HSD, P450 17α, HSL 의 단백질 발현의 의미 있는 변화는 관찰되는 않았다.
간질 세포에서 cholesterol ester 유입에 중요한 요소인 SR-B1 은 cadmium 노출하에서 극적으로 증가하였다. 이러한 결과들을 종합하면, Cadmium 은 P450scc 발현과 HSL 인산화의 억제를 통해 pregnenolone 과 progesterone 생성을 억제한다고 제안할 수 있다.

목차 (Table of Contents)

• Abstract 1
• I. Background 3
• 1. Cadmium 3
• 2. Cadmium-induced cellular toxicity in the testis 4
• 3. Cadmium as an endocrine disruptor 6
• II. Cadmium up-regulates the steroidogenic acute regulatory protein (StAR) gene transcription through phosphorylated-CREB rather than SF-1 in K28 cells 9
• 1. Introduction 9
• 2. Materials and Methods 13
• 3. Results 23
• 3.1 Cadmium increases the levels of StAR mRNA and protein as well as promoter Activity 23
• 3.2 Cadmium-induced StAR expression is involved in PKA and PKC signaling pathways 24
• 3.3 Cadmium regulates expression of the DAX-1 and SF-1 mRNA 25
• 3.4 Cadmium regulates the transcriptional activity of DAX-1 promoter 25
• 3.5 Cadmium regulates the proteins involved in StAR gene transcription 26
• 3.6 Cadmium increases binding of phosphor-CREB, but not SF-1 to StAR Promoter 27
• 3.7 Over-expression of CREB protein enhances cadmium-induced StAR promoter activity 29
• 4.Discussion 49
• III. Cadmium inhibits progesterone production through suppression of P450scc expression in K28 cells 52
• 1. Introduction 52
• 2. Materials and Methods 55
• 3. Results 59
• 3.1 Cadmium decreases the levels of pregnenolone and progesterone 59
• 3.2 Cadmium regulates the proteins involved in steroid hormone production 59
• 3.3 Cadmium regulates the proteins involved in cholesterol uptake 60
• 4.Discussion 68
• IV. References 71
• Abstract in Korean 81