Insulin-like growth factor as a novel stimulator for somatolactin secretion and synthesis in carp pituitary cells via activation of MAPK cascades

Abstract

Jiang Q, Ko WK, Wong AO. Insulin-like growth factor as a novel stimulator for somatolactin secretion and synthesis in carp pituitary cells via activation of MAPK cascades. Am J Physiol Endocrinol Metab 301: E1208 -E1219, 2011. First published August 23, 2011; doi:10.1152/ajpendo.00347.2011, a member of the growth hormone/prolactin family, is a pituitary hormone unique to fish models. Although SL is known to have diverse functions in fish, the mechanisms regulating its secretion and synthesis have not been fully characterized. Using grass carp pituitary cells as a model, here we examined the role of insulin-like growth factor (IGF) in SL regulation at the pituitary level. As a first step, the antisera for the two SL isoforms expressed in the carp pituitary, SL␣ and SL␤, were produced, and their specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the carp pituitary. Western blot using these antisera revealed that grass carp SL␣ and SL␤ could be N-linked glycosylated and their basal secretion and cell content in carp pituitary cells could be elevated by IGF-I and -II treatment. These stimulatory effects occurred with parallel rises in SL␣ and SL␤ mRNA levels, and these SL gene expression responses were not mimicked by insulin but blocked by IGF-I receptor inactivation. In carp pituitary cells, IGF-I and -II could induce rapid phosphorylation of IGF-I receptor, MEK1/2, ERK1/2, MKK3/6, and p38 MAPK; and SL␣ and SL␤ secretion, protein production, and mRNA expression caused by IGF-I and -II stimulation were negated by inactivating MEK1/2 and p38 MAPK. Parallel inhibition of PI3K and Akt, however, were not effective in these regards. These results, taken together, provide evidence that IGF can upregulate SL secretion and synthesis at the pituitary level via stimulation of MAPK-but not PI3K/Akt-dependent pathways.insulin-like growth factor I receptor; mitogen-activated protein kinase; signal transduction; grass carp SOMATOLACTIN (SL), a member of the growth hormone (GH)/ prolactin (PRL) family, is a pituitary hormone unique to fish models (23). Since SL gene could not be identified in the genome databases of tetrapods, including Xenopus, mouse, and human, it is commonly accepted that the gene had been lost in tetrapods during the land invasion of amphibian ancestors (15). To date, two SL isoforms, SL␣ and SL␤, have been reported in bony fish, including zebrafish (53) and grass carp (Ctenopharyngodon idellus) (20). Unlike GH and PRL with expression restricted to pituitary cells in the pars distalis, SL is expressed in periodic acid-Schiff (PAS)-positive cells located in the neurointermediate lobe (NIL) of the fish pituitary (34). PAS staining is commonly used in polysaccharide and glycoprotein detection in biological samples. The sensitivity of SL cells to PAS staining and the presence of N-linked glycosylation sites on SL itself [e.g., in Atlantic cod (39) and carp SL (20)] suggest that SL is a glycoprotein. This idea is supported by the finding that a glycosylated form of SL␣ sensit...

Signal transduction mechanisms for autocrine/paracrine regulation of somatolactin-α secretion and synthesis in carp pituitary cells by somatolactin-α and -β

“…The epitopes detected by these antisera are highly homologous to those of the fish counterparts. These antisera have been used previously in zebrafish (24,31,48), grass carp (17), and rainbow trout (38), confirming that they can cross-react with their respective targets in fish models. After autoradiography, immunoreactive bands were scanned at 8-bit/300 dpi resolution with a Canon LiDE 100 scanner (Canon Japan), saved as TIFF files, and calibrated to an optical density scale using the Image J program (Image J software, NIH).…”

“…Culture medium and pituitary cell lysate from the same well were mixed pro rata to reconstitute the samples for total SL production. These protein samples were resolved in 10% gel by SDS-PAGE, and Western blotting was conducted routinely according to the procedures in a previous description (17). Based on our validation, the antisera for grass carp SL␣ and SL␤ were confirmed to be specific to their respective ligands and had no cross-reactivity for the other SL isoform (17).…”

“…These protein samples were resolved in 10% gel by SDS-PAGE, and Western blotting was conducted routinely according to the procedures in a previous description (17). Based on our validation, the antisera for grass carp SL␣ and SL␤ were confirmed to be specific to their respective ligands and had no cross-reactivity for the other SL isoform (17). In this study, the antisera for SL␣ and SL␤ were used at 1:2,000,000 and 1:1,000,000 dilution, respectively.…”

“…Given that 1) SL is derived from the ancestral GH gene by genome duplication (20) and 2) the localization of SL␣-and SL␤-producing cells is a mosaic pattern in the pars intermedia of grass carp (17), we postulate that SL may retain the intrapituitary regulatory properties of GH to modulate SL secretion and synthesis via autocrine/ paracrine mechanisms. To test the hypothesis, recombinant proteins for the two isoforms of grass carp SL, SL␣ and SL␤, were produced and confirmed to have comparable bioactivity in stimulating pigment aggregation in carp melanophores.…”

“…In a single study using primary culture of sea bass pituitary cells, high doses of leptin could induce SL release, whereas GnRH treatment had no effects in this regard (33). In grass carp pituitary cells, pituitary adenylyl cyclase-activating polypeptide (PACAP), IGF-I, and IGF-II have been identified to be potent stimulators for SL release, synthesis, and gene expression (16,17). These findings suggest that SL gene expression and secretion are controlled by hypothalamic regulation as well as feedback signals from the periphery.…”