Lysophosphatidic acid receptor signaling in mammalian retinal pigment epithelial cells.

PURPOSE: Lysophosphatidic acid (LPA) is a phospholipid growth factor that stimulates proliferation, chemotaxis, cation currents, and K(+) currents in retinal pigment epithelial (RPE) cells. LPA receptor transduction was analyzed in human and rat RPE cells. METHODS: Cells were cultured with standard methods, and signaling pathways were analyzed with a variety of approaches, including whole-cell recording, calcium imaging, and second-messenger assays. RESULTS: LPA-activated nonselective cation currents in rat RPE were blocked by the protein tyrosine kinase (PTK) inhibitor genistein, by the MAP kinase kinase (MEK) inhibitor PD98059, and by loading cells with antibodies to G(alpha(i)/o/t/z). LPA activated the MAP kinase and extracellular signal-related kinase (ERK)-1, and produced a dose-dependent inhibition of cAMP production. LPA stimulated a dose-dependent increase in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was reduced by pretreatment with thapsigargin, suggesting it involves release from intracellular stores. The [Ca(2+)](i) increase was not blocked by ryanodine or the phospholipase C inhibitor U73122. LPA did not stimulate inositol phosphate production. Similar to the cation current, LPA-evoked [Ca(2+)](i) increases were blocked by PD98059 and by loading cells with antibodies to G(alpha(i)/o/t/z). RT-PCR experiments showed the presence of RNA for three LPA receptor subtypes (Edg2, -4, and -7); RNase protection assays showed the strongest expression for Edg2 receptor RNA. CONCLUSIONS: LPA receptors in RPE cells activate pertussis toxin (PTx)-sensitive G proteins that inhibit cAMP accumulation; stimulate MAP kinase which activates a cation current and probably contributes to mitogenesis; and stimulate release of Ca(2+) from intracellular stores that appears independent of IP(3) and ryanodine receptor activation.

Lysophosphatidic acid in airway function and disease.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator and important component of serum. Studies over the past several years which have documented diverse effects of LPA on multiple types of airway cells and which suggest possible involvement of LPA in lung disease are reviewed here. LPA enhances contractility of airway smooth muscle. It also stimulates proliferation of cultured airway smooth muscle cells and exhibits a striking synergism with epidermal growth factor (EGF) for stimulating mitogenesis. Recent studies of the molecular components and signaling pathways mediating synergism are described, including LPA-induced upregulation of EGF receptors and activation of multiple transcription factors by both LPA and EGF. A model for the effects of LPA and EGF on mitogenesis that includes EGF receptor upregulation and synergism between Ras and Rho for activation of the transcription factor AP-1 is presented. LPA stimulates fibronectin secretion and filopodia extension in airway epithelial cells as well as proliferation and collagen gel contraction by lung fibroblasts. A hypothesis for LPA involvement in the airway repair and remodeling, which contribute to the pathology of asthma and other airway diseases, is presented, and future directions for research into the roles of LPA in airway function and disease are suggested.

Lysophosphatidic acid upregulates the epidermal growth factor receptor in human airway smooth muscle cells.

Human airway smooth muscle cells treated with lysophosphatidic acid (LPA) and epidermal growth factor (EGF) exhibit synergistic stimulation of mitogenesis (Ediger TL and Toews ML. J Pharmacol Exp Ther 294: 1076-1082, 2000). The effects of LPA treatment of human airway smooth muscle cells on EGF receptor (EGFR) regulation have now been investigated. LPA treatment for 12-24 h resulted in a twofold increase in (125)I-EGF binding and EGFR protein levels as assessed by Western blot analysis. Competition binding assays indicated single-site binding with an affinity of 3 nM, and the affinity was not changed by LPA treatment. EGFR upregulation was blocked by cycloheximide and actinomycin D, suggesting that LPA influences transcriptional regulation of EGFR expression. Inhibitor studies revealed a prominent role for activation of mitogen-activated protein kinase and p70 ribosomal S6 kinase. Both synergism and EGFR upregulation increased with increased cell density, whereas EGFR expression in control cells decreased. The similar requirements for exposure time, LPA concentrations, and cell confluence suggest that EGFR upregulation may be one contributing factor to the synergistic stimulation of mitogenesis seen with LPA plus EGF.