Signaling mechanisms of growth hormone-releasing hormone receptor in LPS-induced acute ocular inflammation.

Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.

Immunocytochemical localisation of plasma membrane GHRH receptors in human tumours using a novel anti-peptide antibody.

Antagonists of growth hormone releasing hormone (GHRH) directly inhibit the growth of a variety of human neoplasms. However, the plasma membrane receptor mediating these effects has not been immunocytochemically visualised in primary tumour cells. Given that previous attempts using an antibody to the amino-terminal region did not result in the visualisation of plasma membrane receptors, we have developed and characterised an anti-peptide antibody to the carboxy-terminal region 403-422 of the human pituitary GHRH receptor. This sequence is identical to residues 339-358 of splice variant 1 (SV1) of tumoural GHRH receptors. Specificity of the antibody was demonstrated by (1) immunocytochemical staining of GHRH receptor-transfected cells, (2) detection of a broad glycosylated protein band migrating at Mr 50,000-60,000 in Western blots of membranes from human pituitary, and (3) abolition of tissue immunostaining by preadsorbtion of the antibody with its immunising peptide. The distribution of GHRH receptors was investigated in 69 formalin-fixed, paraffin-embedded human tumours showing that GHRH receptors were frequently expressed in breast, ovarian and prostate carcinomas. Immunoreactive GHRH receptors were clearly confined to the plasma membrane and uniformly present on nearly all tumour cells. In Western blots of membranes prepared from human tumours, the anti-GHRH receptor antibody detected a non-glycosylated protein band migrating at Mr 40,000, which corresponds to the expected molecular weight of splice variant 1 of tumoural GHRH receptors. Together, our findings provide direct evidence for the presence of GHRH receptor protein on the plasma membrane of primary human tumour cells. The GHRH receptor visualisation could be of value for a rapid immunohistochemical identification of those tumours which could be a target for diagnostic or therapeutic intervention using GHRH analogues.

Development of a polyclonal antiserum for the detection of the isoforms of the receptors for human growth hormone-releasing hormone on tumors.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various human cancers by multiple mechanisms, which include direct effects on tumor cells through the splice variants (SV) of the GHRH receptor. Our findings suggest that the tumoral protein encoded by SV 1 (SV1) is a likely functional receptor. The aim of this study was to develop a polyclonal antiserum against a polypeptide analog of segment 1-25 of the putative SV1 receptor protein. Rabbits were immunized with [Ala-23]SV1 (1-25)-Tyr-26-Cys-27-NH2 as a hapten, conjugated to BSA or keyhole limpet hemocyanin. The antisera thus generated were evaluated by RIA for binding to the radiolabeled hapten. The specificity and sensitivity of the antisera were studied on xenografts of RL and HT human non-Hodgkin's lymphomas. The sera raised against keyhole limpet hemocyanin-SV1 hapten, showed binding values of 50-75% at a 1:56,000 dilution. In Western blot analyses, the purified polyclonal antibody recognized a specific signal with a molecular mass of approximately 40 kDa in RL and HT lymphomas. This band corresponds to the estimated molecular mass of the GHRH receptor isoform encoded by SV1. RT-PCR and ligand binding studies also revealed the expression of SV1 and the presence of high-affinity binding sites for GHRH on RL and HT tumors. Because the antiserum developed recognizes the tumoral GHRH receptor protein encoded by SV1, it should be of value in various investigations.

Development of a site-directed polyclonal antibody against the pituitary growth hormone-releasing hormone receptor and its use to estimate GHRH receptor concentration in normal and hypothyroid rats.

A site-directed polyclonal antipeptide antibody was generated in rabbits against segment 392-404 of the rat pituitary growth hormone-releasing hormone receptor (GHRH-R), using a multiple antigenic peptide system strategy of immunization. This C-terminal intracellular region of the rat GHRH-R exhibits 85% sequence identity with the human GHRH-R. The purified anti-GHRH-R(392-404) IgGs were characterized in cell lines expressing the human GHRH-R and in rat and human anterior pituitary, using immunoblotting. The polyclonal antibody recognized a 45-kD protein in human GHRH-R-transfected BHK 570 cell membrane preparations but not in wild-type cells. A 45-kD N(alpha)-tagged human GHRH-R was immunodetected with both antitag and anti-GHRH-R antibodies in human GHRH-R-transfected HEK 293 cells. Cross-linking of [(125)I-Tyr(10)]hGHRH(1-44)NH(2) to GHRH-R-transfected BHK cells led to the detection of a major and specific 45-kD radioactive complex. Its probing with the anti-GHRH-R(392-404) IgGs led also to the detection of a 45-kD entity. In rat anterior pituitary homogenates or membrane preparations, immunoblotting led to the detection of 44-, 47- and 65-kD proteins. In human anterior pituitary membrane preparations, immunoblotting led to the detection of 52- and 55-kD proteins. No immunoreactive signal was observed in the rat liver. Cross-linking of [(125)I-Tyr(10)]hGHRH(1-44)NH(2) to rat anterior pituitary homogenates revealed the presence of specific 28-, 47- and 65-kD radioactive complexes. Probing of these radioactive complexes with the anti-GHRH-R(392-404) IgGs resulted in the visualization of 28-, 47- and 65-kD entities and of an additional immunoreactive 44-kD protein. To assess the usefulness of this GHRH-R antibody, estimation of changes in the concentration of rat anterior pituitary GHRH-R was performed by immunoblotting and compared to binding data after a 3-week antithyroid treatment. The treatment known to depress the 2.5- and 4-kb GHRH-R mRNA transcripts by at least 1.7-fold decreased the apparent maximal concentration of high (B(max1)) and low (B(max2)) affinity binding sites by 4.6- and 15.2-fold, respectively, and the 47- and 65-kD GHRH-R proteins by 3.5- and 1. 25-fold, respectively. Altogether, the characteristics of the anti-GHRH-R(392-404) polyclonal antibody indicate that it specifically recognizes the human and rat GHRH-R. It also represents an additional valuable tool to estimate variations of GHRH-Rs in physiopathological conditions known to affect GHRH-R mRNA and/or GHRH binding site concentrations.

Tissue-specific molecular heterogeneity of human growth hormone-releasing hormone receptor protein.

A site-directed anti-peptide antibody (anti-hGHRHRc18) was generated against the cytoplasmic tail of human GHRH receptor. The dissociation constant (Kd) and the antibody binding site (AbT) of anti-hGHRHRc18 were 2.5 nmol/l and 0.54 nmol/l, respectively. In an immunoblotting experiment, affinity-purified anti-hGHRHRc18 specifically recognized a single 50-kDa protein in human pituitary. In a screening of the expression of GHRH receptor protein in extra-pituitary tissues, only human kidney showed a single 52-kDa protein. Our results suggest that the GHRH receptor protein exhibits tissue-specific molecular heterogeneity.