We have previously demonstrated that p38 and extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinases (MAPK) are components of proinflammatory induced cytokine expression in human airway myocytes. The experiments described here further these studies by examining p38 MAPK and NF-κB regulation of cyclooxygenase-2 (COX-2) expression in response to a complex inflammatory stimulus consisting of 10 ng/ml interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and interferon (IFN)-γ. COX-2 expression was induced with this stimulus in a time-dependent manner, with maximal expression seen 12-20 h after treatment. Semiquantitative RT-PCR and immunoblotting experiments demonstrate decreased COX-2 expression following treatment with the p38 MAPK inhibitor SB-203580 (25 μM) or the proteosome inhibitor MG-132 (1 μM). SB-203580 did not affect cytokine-stimulated IκBα degradation, NF-κB nuclear binding activity, or NF-κB-dependent signaling from the COX-2 promoter, indicating that p38 MAPK and NF-κB may affect COX-2 expression via separate signaling pathways. SB-203580, but not MG-132, also increased the initial rate of COX-2 mRNA decay, indicating p38 MAPK, but not NF-κB, participates in the regulation of COX-2 mRNA stability. These findings suggest that although p38 MAPK and NF-κB signaling regulate steady-state levels of COX-2 expression, p38 MAPK additionally affects stability of COX-2 mRNA in cytokine-stimulated human airway myocytes.

The primary function of smooth muscle cells is to contract and alter the stiffness or diameter of hollow organs such as blood vessels, the airways and the gastrointestinal and urogenital tracts. In addition to purely structural functions, smooth muscle cells may play important metabolic roles, particularly in various inflammatory responses. In cell culture, these cells have been shown to be metabolically dynamic, synthesizing and secreting extracellular matrix proteins, glycosaminoglycans and a wide variety of cell-cell signaling proteins, such as interleukins, chemokines and peptide growth factors. Secreted cell signaling proteins participate in the inflammatory response of smooth muscle-containing organs, and some can also stimulate smooth muscle migration, proliferation and contraction. The cellular signaling pathways controlling synthesis of these signaling proteins are similar to those used by cells mediating innate immunity and may contribute to pathogenesis of diverse diseases including atherosclerosis, asthma, inflammatory bowel diseases and preterm labor. Appreciating the role of smooth muscle cells in these diseases may lead to better understanding of the beneficial effects of anti-inflammatory drugs as well as identification of new targets for anti-inflammatory therapy.

Manganese (Mn) is a natural trace metal that is essential for many physiological functions in the human body. Astrocytes in the central nervous system are susceptible reservoirs for Mn accumulation. Estrogen, a steroidal hormone, has been shown to mitigate Mninduced toxicity in cultures of postnatal astrocytes. However, differences in expression/inducibility of glutamate transporters and glutamine synthetase, transmitters, and the natural gonadal steroids and their receptors are known to occur in astrocyte cultures derived from various stages of fetal and postnatal development. Cultures of embryonic (E18) hippocampal astrocytes were examined in this study for the ability of 17 β-estradiol (E2) to protect them from Mn toxicity by up regulating gene expression of a glutamate transporter. Primary rat hippocampal astrocytes were pretreated with β-Estradiol (E2) in vitro and subsequently, Mn sulfate (MnSO 4). The amount of toxic damage to the astrocytes was measured by quantifying glial fibrillary acidic protein (GFAP) with a sandwiched Enzyme-Linked Immunosorbent Assay (ELISA). ELISA analysis indicated Mn exposure at 100 μM, 300 μM, or 600 μM significantly increased GFAP levels. However, E2 concentrations at 10 nM or 30 nM significantly reduced Mn-induced GFAP concentrations at 100 μM. Cells pretreated with 10 nM or 30 nM of E2 significantly lowered GFAP levels. The Water-Soluble Tetrazolium-8 (WST-8) method was utilized to determine cell viability. The WST-8 assay showed that Mn concentrations of 100 μM, 300 μM, or 600 μM significantly reduced the dehydrogenase activity, thereby decreasing the number of viable astrocytes. Enzyme activity with 600 μM of Mn was significantly decreased when compared with 100 μM of Mn, revealing a dose-dependent effect. However, the dehydrogenase activity in cells treated with 600 μM Mn was significantly increased when pretreated with 10 nM of E2. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) was used to measure changes in glutamate transporter-1 gene expression in astrocytes after pretreatment of E2 and subsequently, Mn. PCR analysis showed that when cells were exposed to 300 μM Mn, the GLT-1 gene expression was reduced compared to the control. Data also showed that the GLT-1 mRNA was upregulated in cells pretreated with 10 nM E2. When the cells were pretreated with 10 nM E2 and subsequently, 300 μM Mn, there was an increase in the GLT-1 gene expression. The experimental results indicate that E2 can attenuate some Mn-induced toxicity in E18 astrocytes.