Regulation of corticotropin releasing hormone receptor type 1 messenger RNA level in Y-79 retinoblastoma cells: potential implications for human stress response and immune/inflammatory reaction

We report the regulation of type 1 receptor mRNA in Y-79 human retinoblastoma cells, grown in the absence or presence of pharmacological levels of phorbol esters, forskolin, glucocorticoids and their combinations. To control for inducibility and for assessing the sensitivity of the Y-79 system to glucocorticoids, corticotropin releasing hormone mRNA levels were measured in parallel. All treatments stimulated corticotropin releasing hormone receptor type 1 gene expression relative to baseline. A weak suppression of corticotropin releasing hormone mRNA level was observed during dexamethasone treatment. The cell line expressed ten-fold excess of receptor to ligand mRNA under basal conditions. The findings predict the presence of functional phorbol ester, cyclic AMP and glucocorticoid response elements in the promoter region of corticotropin releasing hormone receptor type 1 gene and support a potential role for its product during chronic stress and immune/inflammatory reaction.


Introduction
Stress response (the organism's ability for adaptive homeostasis) is a major survival resource and an important permissive factor of primal life properties such as growth, reproduction, evolution and adaptation. In mammals, unexpected stimulation or stress, activates the heat shock protein (hsp) system at the cellular level, and the hypothalamic-pituitary-adrenal (HPA) axis at the level of the whole organism. At the molecular level, these two systems communicate through the functional interaction between hsp90 and glucocorticoid receptor (GR). 2 Glucocorticoids are final effectors of the axis, that bind and activate GRs to exert negative feedback regulation at multiple levels of the axis including the proopiomelanocortin (POMC) 3 and corticotropin releasing hormone receptor (CRHR) type 1 genes 4 in the anterior pituitary corticotroph, and the corticotropin releasing hormone (CRH) gene in the paraventricular nucleus (PVN) of the hypothalamus. 5 The CRH system, including the hormone 6 and urocortin v ligands, their two receptor types" type 18-11 expressed primarily in the brain, 12 and type 213-15 with its two splice variant isoforms; z expressed in limited areas of the brain 14 and [3 expressed in peripheral tissues including the duodenum, skeletal muscle, epididymis and perivascular cells of the 1316 heart, and the binding protein (CRHBP), 7 is central coordinator of HPA axis activity and acts in concert with glucocorticoids to maintain whole body homeostasis.
Animal studies revealed highly complex regulation and tissue-specific expression patterns of CRH system components, using a variety of 512.'16 18 21 molecular probes. ' To this end, studies with cell lines expressing components of the CRH system, either naturally or following transfection, provide valuable alternatives to the multiparametric complexity inherent in the animal model systems approach and complement molecular analyses of critical regulatory aspects. [22][23][24][25] The human retinoblastoma cell line Y-79, expresses functional CRHRs. 26  Preparation of DNA probes The probe used for CRHR type 1 mRNA hybridization was a 371 bp long fragment of the 3' non-coding region of the human CRHR type 1 gene that was prepared by direct polymerase chain reaction (PCR) amplification of total human genomic DNA template as described. 28 We used a 785 bp long fragment of CRH cDNA spanning from position 1 (A of the initiator AUG codon) to 785 (in the 3' non-coding region) according to the numbering system of Shibahara et al., 29  The PCR reaction products were fractionated on agarose gels and the 371bp CRHR and 785 bp CRH cDNA fragments were gel purified, quantitated, and P32-1abelled by nick translation, 27 as described. Both probes were labelled to similar specific activities. Preparation and use of human [3 actin cDNA probe for normalization of Northern data, was as previously described. 27 Northern blot analysis Preparation of total cytoplasmic RNA, was performed as previously described. 27 Signal strength was assessed by direct counting wet filters by means of a horizontal radioactivity counting device (Betascope 603 blot analyser, Betagen, Waltham, MA), as described. 27 All analyses were performed in triplicate and the fold inductions obtained were within 10% from the mean. Representative mean total RNA preparations for every experimental condition, were prepared by combining equal (spectrophotometrically) amounts of RNA from each triplicate point, and this report summarizes the Northern data generated by these pooled samples. The quality of pooled total RNA from each treatment group, was assessed by pilot agarose gel electrophoresis using 1 bg RNA per lane, along with RNA size standards. A representative preparation is shown in Fig. 1A. The ethidium bromide staining pattern of the eight different RNAs, suggested that the preparations contained primarily intact .material.
The degree of induction of CRHR type 1 gene expression, was determined by hybridization of the Northern blotted gel containing 20 bg of total RNA per lane, to a 371 bp long fragment of the 3' non-coding region of CRHR type 1 DNA that was prepared by direct PCR amplification of human total genomic DNA template. 2s The results are shown in Induction of CRH mRNA The induction of CRH mRNA by forskolin/PKA, phorbol esters/PKC and glucocorticoid path-ways has been reviewedi 1 The Y-79 cell system expresses detectable amounts of CRH mRNA by Northern blotting (novel finding of this study). We therefore measured CRH mRNA levels in order to assess the responsiveness of the Y-79 cells to the various treatments as well as the relative levels of receptor and ligand and their responses to common inducers. CRH mRNA detection, was made on a duplicate blot to that used for CRHR type 1 mRNA analysis. The probe used for hybridization, was a 785bp DNA segment of the CRH gene including all the protein coding region and a small portion of the 3' non-coding region of exon 2, that was amplified from clone 11, a genomic clone containing the human CRH gene and its flanking regions; as detailed in the Methods section.
Ethidium bromide staining of the electrophoretic pattern of the PCR reaction product, is shown in Fig. 2A. Co-induction of CRH mRNA level, is shown in Fig. 2B  suggests that Y-79 cells are of hypothalamic PVN type and not of placental or central nucleus of the amygdala type, tissues where glucocorticoids upregulate CRH gene expression. 5'18-19 This is also supported by the common glucocorticoid upregulation of CRHR type 4 20 1 mRNA in rat hypothalami and Y-79 cells.

Discussion
We studied the regulation of CRHR type 1 mRNA in Y-79 human retinoblastoma cells by phorbol esters, forskolin, glucocorticoids and their combinations, using CRH gene coexpression as internal control. Given the comparable processing of the filters shown in Figs 1  This correlation weakens the possibility of potential cell line dependent regulatory artifacts and predicts that glucocorticoids may also stimulate human hypothalamic CRHR type 1 gene expression. We cannot exclude the possibility that dexamethasone, a well-known apoptotic factor, may have triggered apoptosis in our system, causing induction of gene expression, not necessarily in line with the HPA regulatory system. However, apoptosis of human lymphocytes, known for their enhanced sensitivity to glucocorticoids, was not seen at the level of the hsp90 system 27-under similar culture conditions, excluding indirectly this possibility in the present Y-79 cellular suspension culture system. In addition to glucocorticoids, forskolin and phorbol esters stimulated both receptor and ligand gene expression, suggesting the presence of functional protein kinase A (PKA) and c(PKC) signal transduction pathways in Y-79 cells, as well as the presence of functional phorbol ester (TRE), cyclic AMP (eRE) and glucocorticoid response elements (GRE) in the promoter region N. C Vamvahopoulos et al.
of CRHR type 1 gene. Combined treatments were used to sustain and confirm the overall primary response trends of CRHR type 1-1igand genes in the Y-79 cell system. Maximal induction for the CRHR type 1 gene was five-fold and for the CRH gene two-fold. These differences may reflect either the relative potencies of the response elements between the two genes, or the presence of novel receptor-specific response element(s). Basally, Y-79 express ten-fold excess of receptor to ligand mRNA, suggesting a potentially inverse autonomous regulation of ligand expression by the receptor. 32 We have not determined whether ligand may also regulate receptor expression in this system.
Our finding of CRHR type 1 mRNA upregulation with concomitant CRH mRNA downregulation by dexamethasone in the Y-79 human retinoblastoma cell line, underscores the regulatory plasticity of the CRH system for the maintenance and/or restoration of homeostasis. The potential biological significance of this observation may be that during classical hypercortisolaemic states such as pregnancy, depression or Cushing's disease, 3 the selective elevation of CRHR type 1 (i.e. in the hypothalamus, 4'2 or other sites) may increase the sensitivity of the CRH system and prime, the organisms homeostatic rebound response. 4 By analogy to immune CRH, 1 local elevation of immune CRHR type 1 in inflammatory sites, such as the arthritic joints of patients with rheumatoid arthritis, may increase the sensitivity of the immune CRH system and prime homeostatic rebound response at the local level. This mechanism, may also account for the beneficial effect of local glucocorticoid administration to inflammatory sites. Further studies on the regulation of CRHR type 2 gene expression by dexamethasone will be needed to support such correlational generalizations.