Kathleen M. Thomas

Transcriptional and post-transcriptional control of lysyl oxidase expression in vascular smooth muscle cells: Effects of TGF-β1 and serum deprivation

Transforming growth factor‐β1 (TGF‐β1) markedly reduced cell proliferation and elevated steady state lysyl oxidase (LO) mRNA 3‐fold in neonatal rat aorta smooth muscle cells cultured in medium containing 10% fetal bovine serum. The increase in LO mRNA was prevented by the presence of cycloheximide, indicative of controlling events at the level of protein synthesis. The basal level of mRNA in cells proliferating in 10% fetal bovine serum in the absence of TGF‐β1 was enhanced 7‐fold upon decreasing growth by shifting to medium containing 0.5% serum. Changes in LO activity paralleled those in LO mRNA. Nuclear run‐on assays revealed that the stimulation of expression in 0.5% serum involved increased gene transcription whereas that caused by TGF‐β1 was mostly post‐transcriptional in origin. LO mRNA was quite labile (t½ approximately 3 h) in 10% serum but was markedly stabilized (t½ > 12 h) by the presence of TGF‐β1 in the 10% serum medium. LO mRNA was also considerably more stable under retarded growth conditions (0.5% serum) in the absence of TGF‐β1. LO promoter activity in luciferase reporter constructs transfected into these cells was low and not significantly affected by the addition of TGF‐β1 to the 10% serum medium but was markedly elevated by shifting from 10 to 0.5% serum in the absence of TGF‐β1. Thus, LO expression is inversely correlated with cell proliferation, and is subject to control at transcriptional and post‐transcriptional levels. TGF‐β1 enhances LO expression in these cells by dramatically stabilizing LO mRNA. J. Cell. Biochem. 65:395–407.

Role of EP2 receptors and cAMP in prostaglandin E2 regulated expression of type I collagen α1, lysyl oxidase, and cyclooxygenase‐1 genes in human embryo lung fibroblasts

In a recent communication, we demonstrated that prostaglandin E2 (PGE2) lowers basal while it ablates interleukin‐1β( (IL‐1β) and transforming growth factor‐β (TGFβ) upregulated lysyl oxidase (LO) mRNA levels. Correspondingly, PGE2 increases cyclooxygenase‐1 (COX1) mRNA in diploid, human embryo lung fibroblasts (IMR90) [Roy et al., 1996]. We now report that these actions by PGE2 are routed through cAMP via the PGE2, EP2 receptor. Among the PGE2 receptor types, the IMR90 predominantly express the EP2 mRNA. These cells also express EP3 and EP4 mRNA at comparatively low levels. Northern blot analyses show that 11‐deoxy PGE1, an EP2/EP4 agonist, emulates the action of PGE2. In a similar manner to PGE2, 11‐deoxy PGE1 decreases basal and TGF‐β induced type I collagen α1 (COL) mRNA, basal and IL‐1β induced LO mRNA while it increases COX1 mRNA. Sulprostone, an EP3/EP1 agonist, has no effect on the expression of these three genes. Forskolin, an adenylate cyclase activator, acts in a very similar manner to PGE2or 11‐deoxy PGE1. It suppresses both basal and TGF‐β induced COL mRNA levels. Both PGE2 and 11‐deoxy PGE1 increase cAMP to a level comparable with forskolin. The role of the EP2 receptor in controlling collagen production is further underscored in the immortalized Rat‐1 fibroblasts, derived from Fischer rat embryos, which do not express detectable EP2 mRNA. In these cells, PGE2 has little effect on COL mRNA level, whereas forskolin increases it. Furthermore, forskolin increases cAMP level in Rat‐1 cells, whereas PGE2 does not. Overall, these results illustrate that much of the PGE2 action on the expression of COL, LO, and COX1 genes is mediated through the EP2 receptor and a subsequent increase in intracellular cAMP. J. Cell. Biochem. 71:254–263, 1998.

Upregulation of lysyl oxidase in vascular smooth muscle cells by cAMP: Role for adenosine receptor activation

Lysyl oxidase (LO) is a key participant in the accumulation of insoluble fibers of elastin and collagen by virtue of its role in the initiation of the covalent cross‐linkages between and within individual molecules comprising these fibers. In view of the essential role played by LO in the accumulation of the fibrotic components of occlusive arterial lesions in atherosclerosis, identification of the signaling molecules which can affect the expression of the LO gene in vascular smooth muscle is of considerable interest. In the present report, we describe evidence for the role of the second messenger, cAMP, in the modulation of the levels of LO in vascular smooth muscle cells. Elevated intracellular cAMP induces the transcription of the LO gene, as revealed by Northern blot analysis and nuclear run on assays. Transient transfection experiments performed with the wild‐type LO promoter and with this promoter mutated at a consensus CREB site, located within the region −100 to −93 base pairs relative to the start of transcription, indicate that cAMP‐induced transcriptional activation is partially due to the presence of this CREB site within the promoter. Activation of stimulatory adenosine receptors in vascular smooth muscle cells with 5′‐N‐ethylcarboxamido adenosine (NECA) increases cAMP, LO mRNA, and enzyme activity. These findings point to the importance of cAMP signaling, potentially initiated by a variety of physiological agents, in the upregulation of LO expression in vascular smooth muscle cells. J. Cell. Biochem. 75:177–185, 1999.

Degradation and repair of elastic fibers in rat lung interstitial fibroblast cultures

Evidence from in vitro and in vivo studies indicates that damaged elastic fibers can be repaired.