Neuroendocrine signaling mechanisms in stress-induced hair loss : The roles of Corticotropin releasing hormone (CRH) and Substance P (SP) in human hair follicles and its cells = 스트레스성 탈모에서 신경내분비 신호 메커니즘 : 인체 모낭과 구성 세포에서의 CRH와 SP의 영향

국문 초록 (Abstract)

최근, 탈모를 포함한 피부 질환들이 스트레스 호르몬 또는 신경내분비 신호에 의해 일어난다는 보고들이 증가되고 있는 추세이다. 대표적인 스트레스 호르몬인 Corticotropin-releasing hormone (CRH)은 시상하부-뇌하수체-부신피질 축 (HPA axis)을 통해 중심적인 스트레스 반응을 규제하는 중요한 역할을 하고 있다. 뿐만 아니라 Substance P (SP) 또한 신체의 스트레스 반응을 매개한다. 이전의 보고에 따르면 CRH 또는 SP는 모낭 내부에서 세포 사멸은 증가시키고, 증식은 억제시킴으로써 모낭의 길이 성장을 억제시킨다는 연구 결과가 있다. 이처럼 스트레스성 탈모에서 CRH 또는 SP의 중요성에 대해서는 많이 보고되고 있지만 세포 수준에서의 자세한 작용기전에 대한 연구는 아직까지 보고된 바 없다.
따라서 본 연구에서는 CRH 또는 SP가 인체 모낭과 그것의 주요 구성 세포에 미치는 영향을 알아보고 천연물로부터 얻어진 물질이 CRH의 영향으로부터 어떻게 보호하는지 연구하고 스트레스성 탈모 개선 소재로써의 효능을 평가하고자 하였다.
CRH와 SP 모두 인체 모낭 조직의 성장을 억제시키고 퇴행기로의 전이를 촉진시켰다. 인체 모낭의 중요한 구성 세포인 모유두세포 (dermal papilla cell, DPC)에서의 영향을 보았을 때, CRH는 세포의 증식을 억제시키고 성장기 관련 마커들의 발현을 감소시켰다. 흥미롭게도, CRH에 의해 POMC, ACTH, cortisol 수치가 증가하는 것을 발견함으로써 모유두세포 단독으로도 충분한 HPA axis가 존재한다는 가능성을 확인하였다. 백두옹 (Pulsatilla chinensis Regel)으로부터 분리된 CRH 수용체 길항제는 CRH에 의한 손상들을 모낭과 모유두세포에서 모두 회복시켜주는 것이 관찰되었다. 또한, SP도 모유두세포의 성장기 관련 마커들의 발현을 조절하였고, 특히, 외모근초세포 (Outer root sheath cell)의 세포 사멸을 유도하였다. 놀랍게도, CRH와 SP는 서로의 수용체 발현을 자극시키는 현상이 관찰되었다.
이러한 결과들을 종합해볼 때, CRH와 SP는 스트레스성 탈모에서 중요한 역할을 하는 인자로 여겨지며, 따라서 각각의 수용체의 길항제는 스트레스성 탈모 치료를 위한 개선소재로써의 사용이 가능할 것으로 생각된다.

다국어 초록 (Multilingual Abstract)

There is a growing interest in the relationship between stress hormones, neuroendocrine signaling, and skin diseases, including hair loss. Corticotropin-releasing hormone (CRH) plays an essential role in regulation of central and local stress responses along the hypothalamic-pituitary-adrenal (HPA) axis. Substance P (SP) is also an important mediator of an acute local and systemic stress responses. Previous study showed CRH or SP inhibited hair growth and induced regression phase (catagen) through regulation of proliferation and apoptosis in hair organ culture system. However, it is not well understood how CRH and SP act on the cells in hair follicle.
The aim of this study is to investigate the effect of CRH and SP on human hair follicles and its cells. In addition, this study is concerned with searching for a CRH receptor antagonist from a library of natural compounds.
Both CRH and SP inhibited hair elongation and induced early catagen transition in ex vivo hair organ culture model. In cultured human dermal papilla cells (DPC), CRH suppressed the proliferation and down-regulated the levels of hair growth-related cytokines. A fully functional HPA axis was demonstrated in cultured hDPCs based on the finding that CRH induced POMC, ACTH, and cortisol expression. CRH receptor antagonists, isolated from Pulsatilla chinensis Regel, restored the CRH-induced modulations in hDPCs. SP modulated the expression of the hair growth-related cytokines in hDPCs, and induced apoptosis in human outer root sheath (ORS) cells. Interestingly, expression of CRH receptors (CRHRs) were increased by SP treatment in cultured hDPCs. Likewise, treatment of CRH induced the expression of NK1, a SP receptor, indicating hormonal and neurogenic stresses may act synergistically to induce hair loss.
These results indicated that both CRH and SP play important role in stress-induced hair loss. Thus, CRH receptor antagonist and/or NK1 antagonist could be a clinical approach for the stress-induced hair loss treatment.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION 4
• 1. Structure and cycling of human hair 4
• 2. CRH and SP: stress signaling 8
• 3. CRH receptor antagonist from P. chinensis 10
• 4. Aim of the study 11
• Ⅱ. MATERIALS AND METHODS 12
• 1. Human hair follicle organ culture 12
• 2. Primary hair follicle cells culture 15
• 3. Hormone treatments 17
• 4. CRH receptor antagonists 17
• 5. Cell viability assessment 19
• 6. Alkaline Phosphatase activity assay 19
• 7. FACS for cell cycle analysis 20
• 8. FACS for apoptosis analysis 20
• 9. Semi quantitative RT-PCR 21
• 10. Western blot analysis 23
• 11. Immunocytochemistry 23
• 12. cAMP assay 25
• 13. Cortisol assay 25
• 14. Statistical analysis 26
• Ⅲ. Action mechanisms of CRH-induced damages in human HFs and its cells 27
• 1. Primary cell culture 27
• 2. CRH, a stress hormone, inhibited hair shaft elongation and induced early catagen transition in human hair follicle organ culture 30
• 3. CRH suppressed the proliferation of hDPCs 32
• 4. hDPCs express CRH receptors (CRHRs) and respond to CRH 35
• 5. Alkaline phosphatase activity was reduced by CRH in cultured hDPCs 37
• 6. CRH down-regulated the expression levels of hair anagen related cytokines in cultured hDPCs 39
• 7. Local HPA axis in cultured hDPCs 41
• 8. The effects of CRH on ORS cells 43
• 9. Discussion on the action mechanism of CRH 46
• Ⅳ. CRH receptor antagonists prepared from Pulsatilla
• chinensis 50
• 1. Cytotoxicity of natural compounds on hDPCs 50
• 2. CRH-induced premature catagen transition was prevented by natural compounds in human hair follicles 52
• 3. Saponin derivatives from P. chinensis protected cultured
• human HFs from effects of CRH 55
• 4. Saponin derivatives from P. chinensis antagonized CRH-induced effects in cultured hDPCs 59
• 5. Discussion on the protective effect of P. chinensis 63
• Ⅴ. Negative effects of Substance P on human HFs and its cells 66
• 1. SP inhibits hair elongation and promotes early catagen transition in human hair follicles 66
• 2. Cultured hDPCs expressed the NK1 receptor 68
• 3. SP suppressed the proliferation of hDPCs through the blocking at G2/M phase entry 68
• 4. SP modulated the hair anagen-related cytokines on cultured hDPCs 72
• 5. SP increased the number of apoptotic cells in ORS cells 72
• 6. The crosstalk between CRH and SP signals 75
• 7. Discussion on the action mechanism of SP 79
• Ⅵ. General discussion and Conclusion 81
• REFERENCES 85
• ABSTRACT IN KOREAN 96