섬유주세포에서 아데노신 A1 길항제에 의한 기질단백분해효소 2의 발현 조절
Regulation of Matrix Metalloproteinase 2 Expression by an Adenosine A1 Agonist in Trabecular Meshwork Cells
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목적: 사람 섬유주세포에서 아데노신 A1 (adenosine A1) 길항제가 금속단백기질분해효소(matrix metalloproteinase 2, MMP-2)의 발현과 조절에 미치는 영향에 대하여 알아보고자 하였다. 대상과 방법: 사람의 섬유주세포를 일차배양한 후 아데노신 A1 길항제인 0.1, 1.0 μM N6-cyclohexyladenosine (CHA)에 2시간 노출시켰다. 이때 아데노신 A1 수용체 저해제인 8-cyclopentyl-1,3-dimethylxanthine (CPT)를 전처치하였다. 금속단백기질분해효소(MMP-2) mRNA의 발현 정도를 측정하였으며, tissue inhibitor of metalloproteinase 2 (TIMP2)와 membrane-type-1 (MT1)-MMP의 단백 활성을 측정하였다. 또한 carboxyfluoresein을 이용하여 섬유주단층세포층의 투과도를 측정하였다. 결과: 1.0 μM CHA는 섬유주단층세포층의 투과도를 농도에 비례하여 유의하게 증가시켰으며(p=0.003), 이는 CPT에 의해 억제되었다. 0.1 μM, 1.0 μM CHA는 MMP-2 mRNA의 발현을 증가시켰으며, 이러한 증가는 CPT에 의해 억제되었다(모두 p<0.05). CHA는 MMP-2 의 활성을 증가시켰으며, TIMP2의 활성을 억제하였고 MT1-MMP 활성을 증가시켰다(모두 p<0.05). 결론: CHA는 섬유주 투과도를 증가시켰다. CHA는 MMP-2의 발현과 활성을 증가시켰으며 TIMP2의 활성을 억제하였고 MT1-MMP 활성을 증가시켰다. 따라서 아데노신 A1 길항제의 작용에서 TIMP2와 MT1-MMP의 활성 변화가 MMP-2의 활성 조절에 관여할 것으로 생각된다.
Abstract
Purpose: We investigated the extent of adenosine A1 agonist-induced expression and regulation of matrix metalloproteinase 2 (MMP-2) synthesis in human trabecular meshwork cells (HTMC). Methods: Primary HTMC cultures were exposed to 0.1 or 1.0 μM N6-cyclohexyladenosine (CHA) for 2 h in the presence or absence of an inhibitor thereof, 8-cyclopentyl-1,3-dimethylxanthine (CPT). The expression level of mRNA encoding MMP-2 was assessed via reverse transcription-polymerase chain reaction, and the levels of tissue inhibitor of metalloproteinase 2 (TIMP2) and membrane-type-1 MMP (MT1-MMP) measured by Western blotting. The permeability of the HTMC monolayer was assessed with the aid of carboxyfluorescein. Results: CHA at 1.0 μM increased the permeability of the HTMC monolayer (p = 0.003) and CHA at both 0.1 and 1.0 μM significantly increased MMP-2 mRNA expression, which was inhibited by co-exposure to CPT (all p < 0.05). CHA increased MMP-2 activity, decreased that of TIMP2, and increased that of MT1-MMP (all p < 0.05). Conclusions: CHA increased the permeability of the HTMC monolayer and increased MMP-2 activity, decreased TIMP2 activity, and increased MT1-MMP activity. Thus, regulation of TIMP2 and MT1-MMP expression may be involved in the adenosine A1 agonist- induced increase in MMP-2 activity.
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