Identification and bioactivity of a granulocyte colony-stimulating factor a homologue from large yellow croaker (Larimichthys crocea).

Granulocyte colony-stimulating factor (GCSF) is a growth factor that drives the proliferation and differentiation of granulocytes and monocytes/macrophages. Currently, two copies of GCSF, named GCSFa and GCSFb, have been identified in teleost fish, but data on the functions and signal pathways of these fish GCSFs are still limited. In the present study, a GCSFa homologue (LcGCSFa) was identified from large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of LcGCSFa is 636 bp long and encodes a protein of 211 amino acids (aa), with a 19-aa signal peptide and a typical IL-6 domain, conserved in fish GCSF sequences. The phylogenetic analysis showed that LcGCSFa clustered with other fish GCSFa homologues. LcGCSFa was constitutively expressed in all tissues tested and significantly up-regulated in head kidney and spleen by Vibrio alginolyticus or poly(I:C). LcGCSFa transcripts were also detected in primary head kidney leucocytes (PKL), primary head kidney macrophages (PKM), and primary head kidney granulocytes (PKG), and significantly up-regulated in PKL and PKG by LPS or poly(I:C). These data indicated that LcGCSFa may be involved in the immune responses induced by bacterium and virus. The recombinant LcGCSFa protein (rLcGCSFa) produced in Pichia pastoris promoted the proliferation of PKL both in vivo and in vitro. Furthermore, rLcGCSFa significantly increased both transcription and phosphorylation levels of the signal transducers and activators of transcription (STAT) proteins (LcSTAT3 and LcSTAT5) in PKL, which are required for the GCSF-dependent proliferation. These results showed that LcGCSFa may promote the proliferation of PKL via the activation of LcSTAT3 and LcSTAT5, suggesting a conserved role across vertebrate GCSFs.