Heavy metal interference with growth hormone signalling in trout hepatoma cells RTH-149.

We have studied the effects of heavy metals (Hg2+, Cu2+, Cd2+) on growth hormone (GH) activation of tyrosine kinase and Ca2+ signaling in the trout (Oncorhynchus mykiss) hepatoma cell line RTH-149. Molecular cloning techniques using primer designed on Oncorhynchus spp. growth hormone receptor (GHR) genes allowed to isolate a highly homologous cDNA fragment from RTH-149 mRNA. Thereafter, cells were analysed by Western blotting or, alternatively, with Ca2+ imaging using fura-2/AM. Exposure of cells to ovine GH alone produced a stimulation of the JAK2/STAT5 pathway and intracellular free Ca2+ variations similar to what has been observed in mammalian models. Cell pre-exposure to Cu2+, Hg2+ or Cd2+ affected cell response to GH by enhancing (Cu2+) or inhibiting (Cd2+) the phosphorylation of JAK2 and STAT5. Heavy metals induced the activation of the MAP kinase p38, and pre-exposure to Hg2+ or Cu2+ followed by GH enhanced the effect of metal alone. Image analysis of fura2-loaded cells indicated that pre-treatment with Hg2+ prior to GH produced a considerable increase of the [Ca2+]i variation produced by either element, while using Cu2+ or Cd2+ the result was similar but much weaker. Data suggest that heavy metals interfere with GH as follows: Hg2+ is nearly ineffective on JAK/STAT and strongly synergistic on Ca2+ signaling; Cu2+ is activatory on JAK/STAT and slightly activatory on Ca2+; Cd2+ is strongly inhibitory on JAK/STAT and slightly activatory on Ca2+; heavy metals could partially activate STAT via p38 independently from GH interaction.