Identification of JAK protein tyrosine kinases as signaling molecules for prolactin. Functional analysis of prolactin receptor and prolactin-erythropoietin receptor chimera expressed in lymphoid cells.

The mechanism of action of prolactin (PRL) was studied in murine lymphoid BAF-3 cells transfected with either the long form of the PRL receptor (PRL-R), or a chimeric receptor consisting of the extracellular domain of the PRL-R and the transmembrane and intracellular domain of the erythropoietin receptor (PRL/EPO-R). PRL sustained normal and long-term proliferation of BAF-3 cells expressing either the PRL-R or the hybrid PRL/EPO-R. Upon [125I]PRL cross-linking, both types of BAF-3 transfectants were shown to express two [125I]PRL cross-linked species differing in size by 20 kDa. These cross-linked complexes, after denaturation, were recognized by antibody against the PRL-R, indicating that they contain the transfected receptor. PRL induced rapid and transient tyrosine phosphorylation of both the PRL-R and the PRL/EPO-R in BAF-3 transfectants. Furthermore, PRL induced rapid tyrosine phosphorylation of the Janus kinase 2 (JAK2) which was already physically associated with the PRL-R or the PRL/EPO-R in the absence of ligand. JAK1 was also associated with PRL-R and PRL/EPO-R in the absence of ligand. However, only in BAF-3 cells expressing the PRL-R does PRL induce rapid and transient tyrosine phosphorylation of JAK1. These results demonstrate that JAK protein tyrosine kinases couple PRL binding to tyrosine phosphorylation and proliferation.     

The Nb2 form of prolactin receptor is able to activate a milk protein gene promoter.

We have recently cloned a cDNA encoding a mutant form of PRL receptor (PRL-R) from Nb2 cells, a PRL-dependent T lymphocyte-derived cell line. This cDNA is identical to the long form of the rat PRL-R, except for a deletion of 594 base pairs in the cytoplasmic domain, resulting in a mature receptor protein of 393 amino acids. Although a segment containing three cytoplasmic regions of moderate to high amino acid sequence identity with members of the PRL/GH receptor family is missing in this receptor form, the region of highest (70%) identity is retained. In the following studies, a homologous functional assay was developed to test the activity of three forms of receptor with respect to their ability to transmit a lactogenic signal. In this system, CHO cells were transiently transfected with a construct containing 2300 base pairs of the 5'-flanking sequence of the rat beta-casein gene fused to the chloramphenicol acetyltransferase (CAT) gene and an expression vector containing the various forms of rat PRL-R cDNA. The transfected cells were grown in serum-free medium in the absence or presence of PRL. In cells transfected with the long form of the PRL-R and beta-casein/CAT construct, a 7.2- +/- 0.9-fold induction (n = 3) of CAT activity was seen when cells were cultured in the presence of 400 ng/ml PRL and 1 micrograms/ml hydrocortisone. This level of stimulation was similar to that observed for the ovine beta-lactoglobulin/CAT construct in which a 5.7- +/- 1.2-fold (n = 3) effect was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of the early immune response against viruses by a teleostean interferon regulatory factor-1 (IRF-1)

We investigated the role of a teleostean interferon regulatory factor-1 (IRF-1) in the regulation of the fish immune system using Japanese flounder, Paralichthys olivaceus, as a model. Fish were intramuscularly vaccinated with a recombinant plasmid expressing the Japanese flounder IRF-1 (JF IRF-1) under the control of the cytomegalovirus immediate/early enhancer (CMV) promoter and were sampled at different days post-immunization. Peripheral blood leukocytes (PBLs) obtained from the JF IRF-1-vaccinated fish during the early stages post-vaccination had significantly elevated levels of nitric oxide (NO) and higher acid phosphatase (AP) activity compared with the control groups. Moreover, supernatants of PBLs obtained from the IRF-1-vaccinated fish contained cytokine-like substances as shown by their protective effect against hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV) in two cell lines, hirame natural embryo (HINAE) cell line and epithelial papillosum of cyprini (EPC) cell line. Relative expression of an anti-viral gene, Mx was highest at the 7th day post-vaccination. Co-injection of JF IRF-1 with a DNA vaccine encoding the major capsid protein (MCP) gene of red seabream iridovirus (RSIV) resulted in elevated serum neutralization antibodies but was not significantly different from that in the fish vaccinated with the DNA vaccine alone. These results suggest that the JF IRF-1 modulates the early immune response in fish and is a potential candidate as genetic adjuvant for vaccination.     

Induction of antiviral state in fish cells by Japanese flounder, Paralichthys olivaceus, interferon regulatory factor-1

Interferon regulatory factor-1 (IRF-1) mediates an antiviral state in cells by regulating the expression of the interferon (IFN-alpha/beta) system. To elucidate the role of IRF-1 in fish during virus infections, we constructed a recombinant plasmid of the Japanese flounder, Paralichthys olivaceus IRF-1 (JF IRF-1) under the control of the cytomegalovirus (CMV) immediate/early enhancer promoter. The antiviral mechanism of JF IRF-1 was studied using transfection experiments in a homologous cell line. Here, we show that cell supernatants obtained from transiently transfected cells enhanced cell viability of a heterologous cell line upon incubation, reduced the titers of hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV), and possessed cytokine-like activity, as shown by their ability to protect cells against virus infections. The supernatants also inhibited the replication of the rhabdoviruses during the early stages of infection as indicated by the reduction of viral titers in the presence of the supernatants obtained from the transfected cells. Further analysis showed that the cell culture supernatants contain cytokine-like substances that possess acid-labile and temperature-resistant properties. These results indicate that JF IRF-1 induces an antiviral state in cells by mediating the production of cytokine-like substances. Thus, JF IRF-1 might be useful as an adjuvant in the development of DNA vaccines against commercially important viral pathogens in Japanese flounder aquaculture.     

A prolactin-dependent immune cell line (Nb2) expresses a mutant form of prolactin receptor.

The Nb2 cell line is a pre-T rat lymphoma that is dependent on prolactin (PRL) for mitogenesis. Two forms of PRL receptor (PRL-R), which differ in the length of their cytoplasmic domains have been identified in different tissues and species. In the present study we have cloned the cDNA and characterized the mitogenic form of PRL-R in Nb2 cells. Polymerase chain reaction amplification of first strand cDNA prepared from Nb2-11C (PRL-dependent) and Nb2-Sp (PRL-independent) cell lines was performed using oligonucleotide primers specific for the binding domain, the short form of the PRL-R, and the cytoplasmic domain of the long form of the PRL-R. These studies indicate that both cell lines express a novel form of PRL-R. A cDNA was isolated from an Nb2-Sp cDNA library, which contains 1446 base pairs identical to the nucleotide sequence of the long form of the rat PRL-R. However, the cDNA sequence is missing 594 base pairs in the cytoplasmic domain compared with the long form of the PRL-R. The cDNA encodes a protein of 393 amino acids, lacking 198 amino acids in the cytoplasmic domain. Scatchard analysis of 125I-labeled ovine prolactin (oPRL) binding to microsomes prepared from transiently transfected COS-7 cells with either PRL-R long form cDNA or Nb2 PRL-R cDNA indicates that the long form of PRL-R binds oPRL with high affinity (K alpha = 8.8 x 10(9) M-1), while the Nb2 PRL-R showed a 3.3-fold increased affinity for PRL (K alpha = 29.1 x 10(9) M-1). In addition, immunoblot analysis of these microsomes using 125I-labeled monoclonal antibody (U6) to the PRL-R demonstrates a Mr of approximately 82,000 for the long form and approximately 62,000 for the Nb2 form of PRL-R. Polymerase chain reaction amplification of genomic DNA prepared from PRL-dependent and -independent cell lines suggests that this form of PRL-R results from a deletion in the PRL-R gene. The identification of a modified long form of PRL-R in the Nb2 cell line should help localize domains of the PRL-R involved in signal transduction and further the investigation of prolactin's role in immune cell proliferation.