Expression and function of vascular estrogen receptors alpha and beta
Sammanfattning: Estrogen receptors (ER) are expressed not only in classical target organs for estrogen like breast and uterus, but also in male reproductive system as well as in non-reproductive organs such as brain, liver and bone. The functions of ER are quite well known in reproductive organs but less information is available regarding their function in vascular tissue. The objective of this thesis was to investigate expression and function of vascular ER. Both ERa and b were expressed in vascular wall as demonstrated by immunocytochemistry and Western blotting. In cultured vascular smooth muscle cells (VSMCs) ERa and b were co-expressed in the nuclei. The selective ERb agonist genistein (100 nM) stimulated vascular protein synthesis determined by [3H]leucine incorporation and this effect was blocked by the pure ER antagonist ICI 182780. DNA synthesis was not affected by genistein treatment, suggesting that genistein-induced stimulation of protein synthesis is not associated with vascular growth. The overall vascular protein expression, determined by SDS PAGE, was identical in mice lacking functional ERb (BERKO), lacking both ERa and b (DERKO) and in wild type (WT) mice. The vascular morphology, media thickness and wall to lumen ratio were not different in DERKO mice compared to WT animals. The force response to noradrenaline was similar in intact aorta from DERKO and WT mice both in the absence and presence of the NOS inhibitor L-NAME and the cyclooxygenase inhibitor indomethacin. Interestingly, the vascular iNOS expression was lower in DERKO mice compared to WT animals, whereas eNOS expression was not affected by lack of both ERa and b. DNA microarray analysis suggested that estrogens down-regulated the vascular expression of a group of inflammatory genes. Real-time PCR showed that 17b-estradiol (E2) attenuated the LPS-induced vascular expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1). The inhibitory effect of E2 was blocked by ICI 182780, suggesting that estrogen reduces vascular recruitment of monocytes/macrophages in an ER-dependent manner. The expression of full-length 66 kDa ERa in cultured VSMCs was increased by E2 treatment within 7 h in the presence of the protein synthesis inhibitor cycloheximide, suggesting that E2 induces a conformational change in the ERa protein. Full-length 54 kDa ERb expression was not affected by E2 treatment in the presence of protein synthesis inhibition. Treatment of VSMCs with the proteasome inhibitior epoxomicin for 3 days caused significant increase in ERa but not ERb expression both in the absence and presence of E2, suggesting that ERa but not ERb is degraded via the ubiquitin-proteasome pathway.
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