Expression and regulation of phospholipase D isoforms in mammalian cell lines.

Phospholipase D (PLD) is activated in mammalian cells in response to diverse stimuli that include growth factors, activators of protein kinase C, and agonists binding to G-protein-coupled receptors. Two forms of mammalian PLD, PLD1 and PLD2, have been identified. Expression of mRNA and protein for PLD1 and PLD2 was analyzed in the following cell lines: A7r5 (rat vascular smooth muscle); EL4 (mouse thymoma); HL-60 (human myeloid leukemia); Jurkat (human leukemia); PC-3 (human prostate adenocarcinoma); PC-12K (rat phaeochromocytoma); and Rat-1 HIR (rat fibroblast). All, with the exception of EL4, express agonist-activated PLD activity. PLD1 is expressed in A7r5, HL-60, PC-3, and Rat-1, while PLD2 is expressed in A7r5, Jurkat, PC12K, PC-3, and Rat-1. Neither isoform is expressed in EL4. Guanine nucleotide-independent PLD activity is present in membranes from all cells expressing PLD2. In PC12K cells, which express only PLD2, treatment with nerve growth factor causes neurite outgrowth and increases expression of PLD2 mRNA and protein within 6-12 h. A corresponding increase is observed in membrane PLD activity and in phorbol-12-myristate-13-acetate (PMA)-stimulated PLD activity in intact cells. These results show that PLD2 can be regulated both pretranslationally and posttranslationally by agonists.

Expression of phospholipase D isoforms in mammalian cells.

Two mammalian isoforms of phospholipase D, PLD1 and PLD2, have recently been characterized at the molecular level. Effects of physiologic agonists on PLD activity in intact cells, as characterized in earlier studies, have generally not been attributed to specific PLD isoforms. Recent work has established that expression of PLD1 and PLD2 varies within tissues and between cell lines. A single cell type can express one, both, or neither isoform, although most cells co-express PLD1 and PLD2. Lymphocytes often lack expression of one or both isoforms of PLD. Relative levels of PLD mRNA expression vary considerably between established cell lines. Expression of transcripts for both PLD1 and PLD2 can be regulated at the transcriptional level by growth and differentiation factors in cultured cells. Thus, it is apparent that the known mammalian PLD isoforms are subject to regulation at the transcriptional level. The available data do not conclusively establish whether PLD1 and PLD2 are the only isoforms responsible for agonist-mediated PLD activation. Further studies of the regulation of expression of PLD isoforms should provide insight into the roles of PLD1 and PLD2 in physiologic responses, and may suggest whether additional forms of PLD remain to be characterized.

Lysophosphatidic acid stimulates phospholipase D activity and cell proliferation in PC-3 human prostate cancer cells.

Phospholipase D (PLD) is activated in mammalian cells in response to a variety of growth factors and may play a role in cell proliferation. Lysophosphatidic acid (LPA) is a bioactive metabolite potentially generated as a result of PLD activation. Two human prostate cancer cell lines, PC-3 and LNCaP, express membrane PLD activity. The effects of LPA on PLD activity and proliferation were examined in PC-3 cells, which express hPLD1a/1b. Phorbol 12-myristate 13-acetate (PMA) induced a prolonged activation of PLD, as detected in both intact cells and membranes. LPA induced a transient activation of PLD that was maximal by 10 minutes. The EC50 for LPA-induced PLD activation was approximately 1 microM. Pertussis toxin did not inhibit activation of PLD by LPA or PMA. Ro-31-8220 and bisindolylmaleimide I, inhibitors of protein kinase C, blocked activation by PLD by both PMA and LPA. PMA-induced activation of PLD did not appear to require translocation of PLDs from cytosol to membrane. LPA stimulated proliferation of PC-3 cells with an EC50 of approximately 0.2 microM; this response was not inhibited by pertussis toxin. Perillyl alcohol, an anti-cancer drug, reversibly inhibited proliferation in response to either serum or LPA but did not inhibit activation of PLD by PMA or LPA. These data establish that LPA activates PLD and stimulates proliferation via Gi-independent pathways in a human prostate cancer cell line.

Peripheral neuropathy as the presenting feature of tyrosinaemia type I and effectively treated with an inhibitor of 4-hydroxyphenylpyruvate dioxygenase.

A 21 month old girl presented with a short history of frequent falls and a right sided foot drop. She went on to suffer recurrent episodes of distal weakness in her arms and legs with hyporeflexia. Electrophysiological studies were consistent with inflammatory demyelinating polyradiculoneuropathy (IDP) and treatment with corticosteroids appeared to lead to an improvement. However, the development of hypertension, evidence of tubulopathy, and hepatomegaly led to re-evaluation. A diagnosis of type I tyrosinaemia was made, based on increased urinary excretion of succinylacetone and decreased activity of fumarylacetoacetase in her cultured skin fibroblasts. A low tyrosine diet did not prevent life-threatening exacerbations of neuropathy but intravenous haemarginate appeared to aid her recovery from one exacerbation. An immediate improvement in strength was seen after starting treatment with 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1,3-cyclohexanedione (NTBC), an inhibitor of 4-hydroxy-phenylpyruvate dioxygenase. A liver transplant was performed but the patient died of immediate postoperative complications. Tyrosinaemia needs to be considered in a child with recurrent peripheral neuropathy because (i) the signs of liver disease and renal tubular dysfunction may be subtle; (ii) acute exacerbations may be life threatening; (iii) specific forms of treatment are available.