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Yefei Pang and Peter Thomas

We have shown progesterone exerts a direct action on vascular smooth muscle cells (VSMCs) to induce relaxation through activation of membrane progesterone receptor alpha (mPRα)-dependent signaling pathways, but information on downstream events is lacking. Progesterone-induced changes in calcium concentrations in human umbilical artery VSMCs through mPRα-dependent signaling pathways and the involvement of Rho/ROCK signaling were investigated. Acute in vitro treatment with progesterone and the selective mPRα agonist 10-ethenyl-19-norprogesterone (Org OD 02-0, 02-0) blocked the rapid prostaglandin F2α-induced calcium increase. This inhibitory progesterone action was prevented by knockdown of mPRα but not by knockdown of the nuclear progesterone receptor, confirming it is mediated through mPRα. The decrease in calcium levels and VSMC relaxation were abolished by treatment with FPL64176 (Ca2+ channel activator), supporting a role for decreased calcium channel activity in this progesterone action. The reduction in calcium was attenuated by pretreatment with pertussis toxin, 8-Bromo-cAMP and forskolin, indicating this progesterone action involves activation of an inhibitory G protein and downregulation of cAMP-dependent signaling. Inhibition of MAPK and Akt signaling with PD98059 and ML-9, respectively, prevented the progesterone-induced calcium concentration decrease and VSMC relaxation. Forskolin decreased progesterone-induced MAPK and Akt phosphorylation which suggests that the cAMP status influences calcium levels indirectly through altering these signaling pathways. Progesterone and 02-0 treatments decreased RhoA activity and ROCK phosphorylation, which suggests that reduced RhoA/ROCK signaling is a component of the mPRα-mediated progesterone actions on VSMCs. The results suggest that progesterone induces VSMC relaxation by reducing cellular calcium levels through mPRα-induced alterations in multiple signaling pathways.

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Yefei Pang and Peter Thomas

Progesterone causes vascular smooth muscle cell relaxation through membrane progesterone receptors (mPRs), which are members of the progestin and adipoQ receptor (PAQR) family, and nuclear PRs (nPRs). However, beneficial vascular effects of progesterone in preventing pre-atherosclerosis and the involvement of mPRs and nPRs remain unclear. The results show short- to long-term treatments with 100 nM progesterone (P4) and specific agonists for mPRs, OD 02-0, and nPRs, R5020, inhibited pre-atherosclerotic events in human umbilical vein endothelial cells (HUVECs), decreasing focal adhesion (FA) by monocytes, FA signaling, HUVEC migration and invasion, and vinculin expression. Progesterone and OD 02-0, but not R5020, inhibited phosphorylation of Src and focal adhesion kinase, critical kinases of FA signaling, within 20 min and migration and invasion of HUVECs and monocyte adhesion after 3 h. These inhibitory P4 and 02-0 effects were attenuated with MAP kinase and Pi3k inhibitors, indicating involvement of these kinases in this mPR-mediated action. However, after 16 h, OD 02-0 was no longer effective in inhibiting FA signaling, while both progesterone and R5020 decreased the activity of the two kinases. Knockdown of receptor expression with siRNA confirmed that mPRα mediates short-term and nPR long-term inhibitory effects of progesterone on FA signaling. Thus, progesterone inhibition of FA signaling and pre-atherosclerosis is coordinated through mPRα and nPRs.

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Yukinori Kazeto, Rie Goto-Kazeto, Peter Thomas, and John M Trant

Membrane-bound progestin receptors (mPR) were recently cloned and characterized as a new class of steroid receptors that transduce cell-signals through alteration of MAP kinase- and cAMP-dependent pathways. To further develop our understanding of this new class of steroid receptors, we characterized the cDNAs and genes of theα, β and γ forms of the channel catfish mPRs (IpmPR). The predicted α and β proteins have 49% sequence identity, whereas they only have 30% and 27% identities, respectively, with the γ form. Furthermore, IpmPRα and IpmPRβ genes have similar structures featuring intronless coding regions, while IpmPRγ gene is composed of 8 exons and 7 introns. The 5′-flanking region of each IpmPR gene differs, but each contains putative transcriptional regulatory elements of factors known to influence reproductive physiology and endocrine disruption, for example, responsive elements for cAMP and steroids and the recognition sites for steroidogenic factor-1 and for the aryl hydrocarbon receptor. The IpmPRα gene was detected in all the tissues tested with relatively greater expression in brain, pituitary, muscle and testis. The expression of IpmPRβ was much lower than that of IpmPRα and the transcript was predominantly observed in brain, pituitary, ovary and testis. In contrast, the IpmPRγ transcript was mainly detected in gill, ventral aorta, intestine, and trunk kidney. In conclusion, all the structural features of the IpmPRs strongly suggest that the closely related α and β forms are distantly related to the γ form. Additionally, regulatory features of the 5′-flanking regions and the differences in tissue-specific expression of each IpmPR gene suggest that they are involved in different endocrine functions in catfish.

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Rene Cortese, Florian Eckhardt, Marianne Volleth, Manfred Wehnert, Uwe Koelsch, Peter Wieacker, and Thomas Brune

Mutations in the LMNA gene cause various phenotypes including partial lipodystrophy, muscular dystrophies, and progeroid syndromes. The specific mutation position within the LMNA sequence can partially predict the phenotype, but the underlying mechanisms for the development of these different phenotypes are still unclear. To investigate whether different DNA methylation patterns contribute to the development of different phenotypes caused by LMNA mutations, we analyzed a panel of ten candidate genes related to fat metabolism, aging, and a tendency to different methylation patterns: CSPG2, ESR1, IGF1R, IGFR2, LMNA, MLH1, RANBP1, RARB, ZMPSTE24, and TGFBR1. We studied two independent families each comprising three individuals affected by familial partial lipodistrophy type 2 (FPLD2). Affected members in each family carried two different mutations of the LMNA gene (R482L and R471G respectively). In addition, we analyzed four progeria patients (2×LMNA/C G608G, 1×LMNA/C S143F, and 1×ZMPSTE24 IVS9-Ex10) and seven healthy adults. The gene encoding retinoic acid receptor B (RARB) showed a higher methylation in all six patients with FPLD2 when compared with the progeria patients with other LMNA mutations as well as the healthy controls (P<0.05). All other investigated genes showed no difference in the methylation patterns between the groups. A drug-induced inhibition of the retinol pathway is discussed as the key pathway for developing HAART-associated lipodystrophy and our data support a possible role of the retinol pathway in the development of lipodystrophy phenotypes.