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Expression of growth hormone-releasing hormone (GHRH) and splice variant of GHRH receptors in normal mouse tissues
Regulatory Peptides 136 (2006) 105 – 108 www.elsevier.com/locate/regpep
Expression of growth hormone-releasing hormone (GHRH) and splice variant of GHRH receptors in normal mouse tissues C. Christodoulou a , A.V. Schally b , I. Chatzistamou c , A. Kondi-Pafiti c , K. Lamnissou d , S. Kouloheri a , A. Kalofoutis a , H. Kiaris a,⁎ a
Department of Biological Chemistry, Medical School, University of Athens, 75 Micras Asias, 115 27 Athens, Greece b Endocrine Polypeptide and Cancer Institute, VA Medical Center, Miami, FL 33125, USA c Department of Pathology, Aretaieion University Hospital, Medical School, University of Athens, Athens, Greece d Department of Biology, University of Athens, Athens, Greece Received 22 December 2005; received in revised form 29 April 2006; accepted 2 May 2006 Available online 16 June 2006
Abstract Growth hormone-releasing hormone (GHRH) stimulates the production and release of growth hormone in the pituitary and induces cell proliferation in a variety of peripheral tissues and tumors. These extrapituitary effects of GHRH are in many cases mediated by a splice variant of GHRH receptor designated SV1 that differs from the pituitary GHRH receptor in a small portion of its amino-terminal region. While SV1 has been detected in several primary tumors and many cancer cell lines its expression in normal tissues remains unclear. In this study we report the results of an immunohistochemical analysis for SV1 and GHRH expression in normal mouse tissues. For the detection of SV1 immunoreactivity we used a polyclonal antiserum against segments 1–25 of the SV1 receptor protein. Mouse heart, colon, lungs, small intestine, stomach and kidneys exhibited increased SV1 immunoreactivity. These tissues were also positive for GHRH expression, however, tissues such as the endometrium were positive only for GHRH and not for SV1 expression. On the contrary, testis were positive for SV1 and not for GHRH expression. These results indicate that SV1 may play a role in normal physiology. © 2006 Elsevier B.V. All rights reserved. Keywords: GHRH; SV1; Receptor; Extrapituitary; Autocrine; Paracrine
1. Introduction Growth hormone-releasing hormone (GHRH) is secreted by the hypothalamus and acts on the pituitary stimulating the production and release of growth hormone [1,2]. The accumulated evidence on the antitumorigenic action of the synthetic analogs of GHRH indicates that besides its neuroendocrine action GHRH can also regulate the rate of cell proliferation by mechanisms other than the stimulation of the pituitary GH-hepatic insulin-like growth factor I axis [1–3]. Such observation points to the direct action of GHRH in peripheral tissues [1–3]. These effects have been shown to be mediated, at least in part, by a protein encoded by SV1, a splice variant of GHRH receptor that has been detected in various primary tumors and cell lines [4,5]. SV1 differs from the pituitary GHRH receptor ⁎ Corresponding author. Tel./fax: +30 210 7462695. E-mail address: [email protected] (H. Kiaris). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.05.001
in a small portion of its amino-terminal in the extracellular part of the receptor. Functional assays indicated that SV1 encodes for a receptor that can bind to GHRH and upon binding transduces signals that are capable of stimulating the rate of cell proliferation [5]. Furthermore, it has been demonstrated that SV1, besides being ligand-dependent, also possesses ligand-independent activity, that is consistent with the stimulation of cell proliferation in the absence of ligand binding [6]. It appears however that in the presence of ligand, namely GHRH, the effects of SV1 can be potentiated [6]. Despite the fact that much information on the functional properties and the expression of SV1 in several neoplastic tissues is available [7–9], its pattern of expression in normal tissues remains unclear. Such data would be of particular interest not only because it could outline a function for GHRH and SV1 in the periphery, but also because it might identify tissues serving as models to examine the extrapituitary role of GHRH. Thus we have studied the expression of both GHRH and SV1 in a bank of normal mouse
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Table 1 Anti-GHRH and anti-SV1 immunoreactivity in normal mouse tissues Tissue
GHRH
SV1
Positivity Cell type
Positivity Cell type
Endometrium +
Endometrial gland cells
− −
Muscle Salivary glands Liver Heart
+ +
Colon
±
Lung
++
Spleen Kidney
− +
Urinary bladder Small intestine (jejunum) Small intestine (ileum) Stomach
−
Testis
−
+
+
++
− − −
Hepatocytes Diffusely myocardial cells Apical border cells Mostly alveolar cells Predominantly collective duct cells
++ +
Hepatocytes Diffusely myocardial cells
+
Apical border cells
+
Alveolar cells
− +
Predominantly collective duct cells
− Mostly intestinal villi cells Mostly intestinal villi cells Gastric gland cells
+
Mostly intestinal villi cells
++
Mostly intestinal villi cells
+
Gastric gland cells
++
Mostly spermatocytes
tissues by immunohistochemistry. Our results indicate that GHRH and SV1 are detectable in peripheral tissues and in most of the cases they are co-expressed. 2. Materials and methods 2.1. Specimens
evaluate expression of SV1 in mouse tissues, in preliminary experiments, the specificity of the corresponding antibody has been evaluated by pre-incubating the antiserum with increasing concentrations of the original peptide used for the immunization of rabbits, as described previously [11]. These experiments showed that immunopositivity was being abolished by this treatment, arguing in favor of the specificity of our results. Negative controls in which the primary antibody has been omitted have also been included in the analysis (data not shown). Specimens were evaluated for positive staining and classified according to the percentage of positive cells, into the following categories: 1–10%, ±; 11–30%, +; 31–70%, ++; 71–100%, +++. Images shown were obtained by Pro-image Analysis Software (Media Cybernetics, Inc, MD). 3. Results and discussion A summary of the results on the evaluation of the expression of GHRH and its receptor's splice variant SV1 in normal mouse tissues is shown in Table 1. In accordance with previous studies that have reported the identification of GHRH in normal peripheral tissues [12–16], we have detected GHRH immunoreactivity in 10 out of 14 tissues tested (Fig. 1). Immunoreactivity ranged from sparse as in the colon to intense, as in the lungs. This finding indicates that these GHRH-positive tissues either express or accumulate GHRH that is expressed in other tissues. Immunoreactivity was cytoplasmic in all cases, which is consistent with the expression of GHRH by these tissues or can also be due to the internalization of this peptide hormone and its subsequent intracellular degradation. In previous studies in which GHRH immunoreactivity was evaluated in malignant endometrial tissue, positive staining was also detected in nucleus [17]. The absence of nuclear immunopositivity in the present study which involved normal tissues only, may indicate that the presence of GHRH in the nucleus of cancer cells is due to its mislocalization, as has already been suggested before [17]. The major receptor for extrapituitary GHRH identified to date is SV1 which differs from the pituitary GHRH receptor in a small portion of its amino-terminus [4]. In the present study SV1 expression was detected in several tissues that also expressed GHRH
The tissues were obtained from 10 to 12 week old male and female wild type mice (mixed C57BL6 and C3H genetic background) originally obtained from Jackson Laboratories (Bar Arbor, ME). Tissues were subsequently fixed in 10% formalin and paraffin-embedded by using standard procedures. 2.2. Immunohistochemistry Four (4) μm thick sections from representative paraffin-embedded blocks were collected onto poly-L-lysine-coated slides and stained for GHRH and SV1 antigens. The immunohistochemical detection of GHRH was carried out with the rabbit anti-GHRH SV95 [10] polyclonal antibody, diluted with 1× Phosphate Buffer Saline (PBS) at 1:100, using the Kwik-DAB kit (ThermoShandon, Pittsburgh, PA, USA) according to manufacturer's instructions. The immunohistochemical detection of SV1 was performed with the rabbit anti-SV1 polyclonal antibody 2317/5 diluted with 1× PBS at 1:104 [11]. Since this is the first time this antibody is being used to
Fig. 1. Representative microphotographs of GHRH immunoreactivity in normal lung tissue. Immunopositivity is indicated by the brown staining (arrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
C. Christodoulou et al. / Regulatory Peptides 136 (2006) 105–108
including small intestine, liver, heart colon, lung, kidney, and stomach (Fig. 2). Testis also expressed SV1 but not GHRH while endometrium and uterus were positive for GHRH only. In all cases immunoreactivity was cytoplasmic. When these results are taken together the tissues examined can be divided into 3 groups according to the status of combined GHRH and SV1 expression. Group A includes tissues that express both GHRH and SV1, group B involving tissues expressing GHRH only and not SV1, group C that includes tissues expressing only SV1 and not GHRH, and group D in which tissues are negative for both antigens examined. Group A tissues most likely correspond to tissues in which an autocrine loop between GHRH and its receptor is operational since the co-localization of both the ligand and its receptor was detected. This group includes the majority of the tissues tested (8 out of 14) and for them an important physiological role for GHRH is possible. Of particular interest are the tissues of group B that express only GHRH and not SV1. Assuming that the presence of GHRH in these tissues is not coincidental but physiologically important, two possibilities may explain this finding: The first possibility is consistent with the paracrine/endocrine action of this neurohormone according to which the tissue of production and the target tissue are different. The alternative possibility is that the extrapituitary effects of GHRH are mediated not only by SV1 but by other receptor(s) as well. Thus, in some tissues GHRH may act through SV1 while in others via other receptors. Indeed, most likely candidates for these receptors are the members of the VIP/
107
PACAP receptor superfamily that share considerable homology with the GHRH receptor, while other, not yet identified receptors may also mediate the extrapituitary effects of GHRH [17]. Group C tissues that express only SV1 and not GHRH, such as the testis, may operate through the previously reported ligand-independent activity of SV1 [5] or may be targeted by the systemic GHRH. By using a more sensitivity methodology, such as RT-PCR and in situ hybridization, the detection of GHRH and SV1 in tissues that scored negative in this study is possible, and thus our results should be considered with some caution. However, it has to be noted that only detectable expression of GHRH and SV1 should be viewed as physiologically important. Indeed, in an earlier study, expression of GHRH and GHRH receptor in rat tissues was evaluated by the highly sensitive RT-PCR coupled by Southern blot hybridization [18]. Widespread expression of both the ligand and receptor was found in various extrapituitary tissues which could be due, at least in part to specific SV1 expression. Collectively our results indicate that GHRH and its receptor SV1 are expressed in normal mouse tissues implying an extrapituitary role for this neurohormone under physiological conditions. The precise mechanism of action of GHRH in peripheral tissues merits further investigations. Acknowledgments This study was supported by grant Pythagoras II from the Ministry of Education (to S.K).
Fig. 2. Representative microphotographs of SV1 immunoreactivity in normal mouse tissues. The tissue is indicated at the top-left corner of each microphotograph. Immunopositivity is indicated by the brown staining (arrow). Liver shows quite intense and uniform cytoplasmic staining. Salivary glands that are negative are also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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References [1] Kiaris H, Schally AV, Kalofoutis A. Extra-pituitary effects of growth hormone-releasing hormone. Vitam Horm 2005;70:1–24. [2] Schally AV, Comaru-Schally AM, Nagy A, Kovacs M, Szepeshazi K, Plonowski A, Varga JL, Halmos G. Hypothalamic hormones and cancer. Front Neuroendocrinol 2001;22:248–91. [3] Kiaris H, Koutsilieris M, Kalofoutis A, Schally AV. Growth hormonereleasing hormone (GHRH) and extra-pituitary tumorigenesis: therapeutic and diagnostic applications of GHRH antagonists (review). Exp Opin Invest Drugs 2003;12:1385–94. [4] Rekasi Z, Czompoly T, Schally AV, Halmos G. Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers. Proc Natl Acad Sci U S A 2000;97:10561–6. [5] Kiaris H, Schally AV, Busto R, Halmos G, Artavanis-Tsakonas S, Varga JL. Expression of splice variant for GHRH receptor SV1 mediates mitogenic effects in 3T3 fibroblasts. Proc Natl Acad Sci U S A 2002;99:196–200. [6] Kiaris H, Chatzistamou I, Schally AV, Halmos G, Varga JL, Koutselini H, Kalofoutis A. Ligand-dependent and -independent effects of GHRH receptor splice variant 1. Proc Natl Acad Sci U S A 2003;100:9512–7. [7] Busto R, Schally A, Braczkowski R, Plonowski A, Krupa M, Groot K, Armatis P, Varga J. Expression of mRNA for growth hormone-releasing hormone and splice variants of GHRH receptors in human malignant bone tumors. Regul Pept 2002;108:47–53. [8] Busto R, Schally AV, Varga JL, Garcia-Fernandez MO, Groot K, Armatis P, Szepeshazi K. The expression of growth hormone-releasing hormone (GHRH) and splice variants of its receptor in human gastroenteropancreatic carcinomas. Proc Natl Acad Sci U S A 2002;99:11866–71. [9] Garcia-Fernandez MO, Schally AV, Varga JL, Groot K, Busto R. The expression of growth hormone-releasing hormone (GHRH) and its receptor splice variants in human cancer cell lines; the evaluation of signaling mechanisms in the stimulation of cell proliferation. Breast Cancer Res Treat 2003;77:15–26.
[10] Groot K, Csernus VJ, Pinski J, Zsigo J, Rekasi Z, Zarandi M, et al. Development of a radioimmunoassay for some agonists of growth hormone-releasing hormone. Int J Pept Protein Res 1993;41:162–8. [11] Toller GL, Horvath JE, Schally AV, Halmos G, Varga JL, Groot K, Chism D, Zarandi M. Development of a polyclonal antiserum for the detection of the isoforms of the receptors for human growth hormone-releasing hormone on tumors. Proc Natl Acad Sci U S A 2004;101:15160–5. [12] Margioris NM, Brockmann G, Bohler Jr HCL, Grino M, Vamvakopoulos N, Chrousos GP. Expression and localization of growth hormone-releasing hormone messenger ribonucleic acid in rat placenta: in vitro secretion and regulation of its peptide product. Endocrinology 1990;126:151–8. [13] Moretti C, Fabbri A, Gnessi L, Bonifacio V, Bolotti M, Arizzi M, Nazziocone Q, Spera G. Immunohistochemical localization of growth hormone-releasing hormone in human gonads. J Endocrinol Invest 1990;13:301–5. [14] Stephanou A, Knight RA, Lightman SL. Production of growth hormonereleasing hormone-like peptide and its mRNA by human lymphocytes. Neuroendocrinology 1991;53:628–33. [15] Bagnato A, Moretti C, Ohnishi J, Frajese G, Catt KJ. Expression of the growth hormone-releasing hormone and its peptide product in the rat ovary. Endocrinology 1992;130:1097–102. [16] Chatzistamou I, Schally AV, Pafiti A, Kiaris H, Koutselini H. Expression of growth hormone-releasing hormone (GHRH) in human primary endometrial carcinomas. Eur J Endocrinol 2002;147:381–6. [17] Sherwood NM, Krueckl SL, McRory JE. The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocrine Rev 2000;21:619–70. [18] Matsubara S, Sato M, Mizobuchi M, Niimi M, Takahara J. Differential gene expression of growth hormone (GH)-releasing hormone (GRH) and GRH receptor in various rat tissues. Endocrinology 1995;136:4147–50.
Expression of growth hormone-releasing hormone (GHRH) and splice variant of GHRH receptors in normal mouse tissues C. Christodoulou a , A.V. Schally b , I. Chatzistamou c , A. Kondi-Pafiti c , K. Lamnissou d , S. Kouloheri a , A. Kalofoutis a , H. Kiaris a,⁎ a
Department of Biological Chemistry, Medical School, University of Athens, 75 Micras Asias, 115 27 Athens, Greece b Endocrine Polypeptide and Cancer Institute, VA Medical Center, Miami, FL 33125, USA c Department of Pathology, Aretaieion University Hospital, Medical School, University of Athens, Athens, Greece d Department of Biology, University of Athens, Athens, Greece Received 22 December 2005; received in revised form 29 April 2006; accepted 2 May 2006 Available online 16 June 2006
Abstract Growth hormone-releasing hormone (GHRH) stimulates the production and release of growth hormone in the pituitary and induces cell proliferation in a variety of peripheral tissues and tumors. These extrapituitary effects of GHRH are in many cases mediated by a splice variant of GHRH receptor designated SV1 that differs from the pituitary GHRH receptor in a small portion of its amino-terminal region. While SV1 has been detected in several primary tumors and many cancer cell lines its expression in normal tissues remains unclear. In this study we report the results of an immunohistochemical analysis for SV1 and GHRH expression in normal mouse tissues. For the detection of SV1 immunoreactivity we used a polyclonal antiserum against segments 1–25 of the SV1 receptor protein. Mouse heart, colon, lungs, small intestine, stomach and kidneys exhibited increased SV1 immunoreactivity. These tissues were also positive for GHRH expression, however, tissues such as the endometrium were positive only for GHRH and not for SV1 expression. On the contrary, testis were positive for SV1 and not for GHRH expression. These results indicate that SV1 may play a role in normal physiology. © 2006 Elsevier B.V. All rights reserved. Keywords: GHRH; SV1; Receptor; Extrapituitary; Autocrine; Paracrine
1. Introduction Growth hormone-releasing hormone (GHRH) is secreted by the hypothalamus and acts on the pituitary stimulating the production and release of growth hormone [1,2]. The accumulated evidence on the antitumorigenic action of the synthetic analogs of GHRH indicates that besides its neuroendocrine action GHRH can also regulate the rate of cell proliferation by mechanisms other than the stimulation of the pituitary GH-hepatic insulin-like growth factor I axis [1–3]. Such observation points to the direct action of GHRH in peripheral tissues [1–3]. These effects have been shown to be mediated, at least in part, by a protein encoded by SV1, a splice variant of GHRH receptor that has been detected in various primary tumors and cell lines [4,5]. SV1 differs from the pituitary GHRH receptor ⁎ Corresponding author. Tel./fax: +30 210 7462695. E-mail address: [email protected] (H. Kiaris). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.05.001
in a small portion of its amino-terminal in the extracellular part of the receptor. Functional assays indicated that SV1 encodes for a receptor that can bind to GHRH and upon binding transduces signals that are capable of stimulating the rate of cell proliferation [5]. Furthermore, it has been demonstrated that SV1, besides being ligand-dependent, also possesses ligand-independent activity, that is consistent with the stimulation of cell proliferation in the absence of ligand binding [6]. It appears however that in the presence of ligand, namely GHRH, the effects of SV1 can be potentiated [6]. Despite the fact that much information on the functional properties and the expression of SV1 in several neoplastic tissues is available [7–9], its pattern of expression in normal tissues remains unclear. Such data would be of particular interest not only because it could outline a function for GHRH and SV1 in the periphery, but also because it might identify tissues serving as models to examine the extrapituitary role of GHRH. Thus we have studied the expression of both GHRH and SV1 in a bank of normal mouse
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Table 1 Anti-GHRH and anti-SV1 immunoreactivity in normal mouse tissues Tissue
GHRH
SV1
Positivity Cell type
Positivity Cell type
Endometrium +
Endometrial gland cells
− −
Muscle Salivary glands Liver Heart
+ +
Colon
±
Lung
++
Spleen Kidney
− +
Urinary bladder Small intestine (jejunum) Small intestine (ileum) Stomach
−
Testis
−
+
+
++
− − −
Hepatocytes Diffusely myocardial cells Apical border cells Mostly alveolar cells Predominantly collective duct cells
++ +
Hepatocytes Diffusely myocardial cells
+
Apical border cells
+
Alveolar cells
− +
Predominantly collective duct cells
− Mostly intestinal villi cells Mostly intestinal villi cells Gastric gland cells
+
Mostly intestinal villi cells
++
Mostly intestinal villi cells
+
Gastric gland cells
++
Mostly spermatocytes
tissues by immunohistochemistry. Our results indicate that GHRH and SV1 are detectable in peripheral tissues and in most of the cases they are co-expressed. 2. Materials and methods 2.1. Specimens
evaluate expression of SV1 in mouse tissues, in preliminary experiments, the specificity of the corresponding antibody has been evaluated by pre-incubating the antiserum with increasing concentrations of the original peptide used for the immunization of rabbits, as described previously [11]. These experiments showed that immunopositivity was being abolished by this treatment, arguing in favor of the specificity of our results. Negative controls in which the primary antibody has been omitted have also been included in the analysis (data not shown). Specimens were evaluated for positive staining and classified according to the percentage of positive cells, into the following categories: 1–10%, ±; 11–30%, +; 31–70%, ++; 71–100%, +++. Images shown were obtained by Pro-image Analysis Software (Media Cybernetics, Inc, MD). 3. Results and discussion A summary of the results on the evaluation of the expression of GHRH and its receptor's splice variant SV1 in normal mouse tissues is shown in Table 1. In accordance with previous studies that have reported the identification of GHRH in normal peripheral tissues [12–16], we have detected GHRH immunoreactivity in 10 out of 14 tissues tested (Fig. 1). Immunoreactivity ranged from sparse as in the colon to intense, as in the lungs. This finding indicates that these GHRH-positive tissues either express or accumulate GHRH that is expressed in other tissues. Immunoreactivity was cytoplasmic in all cases, which is consistent with the expression of GHRH by these tissues or can also be due to the internalization of this peptide hormone and its subsequent intracellular degradation. In previous studies in which GHRH immunoreactivity was evaluated in malignant endometrial tissue, positive staining was also detected in nucleus [17]. The absence of nuclear immunopositivity in the present study which involved normal tissues only, may indicate that the presence of GHRH in the nucleus of cancer cells is due to its mislocalization, as has already been suggested before [17]. The major receptor for extrapituitary GHRH identified to date is SV1 which differs from the pituitary GHRH receptor in a small portion of its amino-terminus [4]. In the present study SV1 expression was detected in several tissues that also expressed GHRH
The tissues were obtained from 10 to 12 week old male and female wild type mice (mixed C57BL6 and C3H genetic background) originally obtained from Jackson Laboratories (Bar Arbor, ME). Tissues were subsequently fixed in 10% formalin and paraffin-embedded by using standard procedures. 2.2. Immunohistochemistry Four (4) μm thick sections from representative paraffin-embedded blocks were collected onto poly-L-lysine-coated slides and stained for GHRH and SV1 antigens. The immunohistochemical detection of GHRH was carried out with the rabbit anti-GHRH SV95 [10] polyclonal antibody, diluted with 1× Phosphate Buffer Saline (PBS) at 1:100, using the Kwik-DAB kit (ThermoShandon, Pittsburgh, PA, USA) according to manufacturer's instructions. The immunohistochemical detection of SV1 was performed with the rabbit anti-SV1 polyclonal antibody 2317/5 diluted with 1× PBS at 1:104 [11]. Since this is the first time this antibody is being used to
Fig. 1. Representative microphotographs of GHRH immunoreactivity in normal lung tissue. Immunopositivity is indicated by the brown staining (arrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
C. Christodoulou et al. / Regulatory Peptides 136 (2006) 105–108
including small intestine, liver, heart colon, lung, kidney, and stomach (Fig. 2). Testis also expressed SV1 but not GHRH while endometrium and uterus were positive for GHRH only. In all cases immunoreactivity was cytoplasmic. When these results are taken together the tissues examined can be divided into 3 groups according to the status of combined GHRH and SV1 expression. Group A includes tissues that express both GHRH and SV1, group B involving tissues expressing GHRH only and not SV1, group C that includes tissues expressing only SV1 and not GHRH, and group D in which tissues are negative for both antigens examined. Group A tissues most likely correspond to tissues in which an autocrine loop between GHRH and its receptor is operational since the co-localization of both the ligand and its receptor was detected. This group includes the majority of the tissues tested (8 out of 14) and for them an important physiological role for GHRH is possible. Of particular interest are the tissues of group B that express only GHRH and not SV1. Assuming that the presence of GHRH in these tissues is not coincidental but physiologically important, two possibilities may explain this finding: The first possibility is consistent with the paracrine/endocrine action of this neurohormone according to which the tissue of production and the target tissue are different. The alternative possibility is that the extrapituitary effects of GHRH are mediated not only by SV1 but by other receptor(s) as well. Thus, in some tissues GHRH may act through SV1 while in others via other receptors. Indeed, most likely candidates for these receptors are the members of the VIP/
107
PACAP receptor superfamily that share considerable homology with the GHRH receptor, while other, not yet identified receptors may also mediate the extrapituitary effects of GHRH [17]. Group C tissues that express only SV1 and not GHRH, such as the testis, may operate through the previously reported ligand-independent activity of SV1 [5] or may be targeted by the systemic GHRH. By using a more sensitivity methodology, such as RT-PCR and in situ hybridization, the detection of GHRH and SV1 in tissues that scored negative in this study is possible, and thus our results should be considered with some caution. However, it has to be noted that only detectable expression of GHRH and SV1 should be viewed as physiologically important. Indeed, in an earlier study, expression of GHRH and GHRH receptor in rat tissues was evaluated by the highly sensitive RT-PCR coupled by Southern blot hybridization [18]. Widespread expression of both the ligand and receptor was found in various extrapituitary tissues which could be due, at least in part to specific SV1 expression. Collectively our results indicate that GHRH and its receptor SV1 are expressed in normal mouse tissues implying an extrapituitary role for this neurohormone under physiological conditions. The precise mechanism of action of GHRH in peripheral tissues merits further investigations. Acknowledgments This study was supported by grant Pythagoras II from the Ministry of Education (to S.K).
Fig. 2. Representative microphotographs of SV1 immunoreactivity in normal mouse tissues. The tissue is indicated at the top-left corner of each microphotograph. Immunopositivity is indicated by the brown staining (arrow). Liver shows quite intense and uniform cytoplasmic staining. Salivary glands that are negative are also shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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References [1] Kiaris H, Schally AV, Kalofoutis A. Extra-pituitary effects of growth hormone-releasing hormone. Vitam Horm 2005;70:1–24. [2] Schally AV, Comaru-Schally AM, Nagy A, Kovacs M, Szepeshazi K, Plonowski A, Varga JL, Halmos G. Hypothalamic hormones and cancer. Front Neuroendocrinol 2001;22:248–91. [3] Kiaris H, Koutsilieris M, Kalofoutis A, Schally AV. Growth hormonereleasing hormone (GHRH) and extra-pituitary tumorigenesis: therapeutic and diagnostic applications of GHRH antagonists (review). Exp Opin Invest Drugs 2003;12:1385–94. [4] Rekasi Z, Czompoly T, Schally AV, Halmos G. Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers. Proc Natl Acad Sci U S A 2000;97:10561–6. [5] Kiaris H, Schally AV, Busto R, Halmos G, Artavanis-Tsakonas S, Varga JL. Expression of splice variant for GHRH receptor SV1 mediates mitogenic effects in 3T3 fibroblasts. Proc Natl Acad Sci U S A 2002;99:196–200. [6] Kiaris H, Chatzistamou I, Schally AV, Halmos G, Varga JL, Koutselini H, Kalofoutis A. Ligand-dependent and -independent effects of GHRH receptor splice variant 1. Proc Natl Acad Sci U S A 2003;100:9512–7. [7] Busto R, Schally A, Braczkowski R, Plonowski A, Krupa M, Groot K, Armatis P, Varga J. Expression of mRNA for growth hormone-releasing hormone and splice variants of GHRH receptors in human malignant bone tumors. Regul Pept 2002;108:47–53. [8] Busto R, Schally AV, Varga JL, Garcia-Fernandez MO, Groot K, Armatis P, Szepeshazi K. The expression of growth hormone-releasing hormone (GHRH) and splice variants of its receptor in human gastroenteropancreatic carcinomas. Proc Natl Acad Sci U S A 2002;99:11866–71. [9] Garcia-Fernandez MO, Schally AV, Varga JL, Groot K, Busto R. The expression of growth hormone-releasing hormone (GHRH) and its receptor splice variants in human cancer cell lines; the evaluation of signaling mechanisms in the stimulation of cell proliferation. Breast Cancer Res Treat 2003;77:15–26.
[10] Groot K, Csernus VJ, Pinski J, Zsigo J, Rekasi Z, Zarandi M, et al. Development of a radioimmunoassay for some agonists of growth hormone-releasing hormone. Int J Pept Protein Res 1993;41:162–8. [11] Toller GL, Horvath JE, Schally AV, Halmos G, Varga JL, Groot K, Chism D, Zarandi M. Development of a polyclonal antiserum for the detection of the isoforms of the receptors for human growth hormone-releasing hormone on tumors. Proc Natl Acad Sci U S A 2004;101:15160–5. [12] Margioris NM, Brockmann G, Bohler Jr HCL, Grino M, Vamvakopoulos N, Chrousos GP. Expression and localization of growth hormone-releasing hormone messenger ribonucleic acid in rat placenta: in vitro secretion and regulation of its peptide product. Endocrinology 1990;126:151–8. [13] Moretti C, Fabbri A, Gnessi L, Bonifacio V, Bolotti M, Arizzi M, Nazziocone Q, Spera G. Immunohistochemical localization of growth hormone-releasing hormone in human gonads. J Endocrinol Invest 1990;13:301–5. [14] Stephanou A, Knight RA, Lightman SL. Production of growth hormonereleasing hormone-like peptide and its mRNA by human lymphocytes. Neuroendocrinology 1991;53:628–33. [15] Bagnato A, Moretti C, Ohnishi J, Frajese G, Catt KJ. Expression of the growth hormone-releasing hormone and its peptide product in the rat ovary. Endocrinology 1992;130:1097–102. [16] Chatzistamou I, Schally AV, Pafiti A, Kiaris H, Koutselini H. Expression of growth hormone-releasing hormone (GHRH) in human primary endometrial carcinomas. Eur J Endocrinol 2002;147:381–6. [17] Sherwood NM, Krueckl SL, McRory JE. The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocrine Rev 2000;21:619–70. [18] Matsubara S, Sato M, Mizobuchi M, Niimi M, Takahara J. Differential gene expression of growth hormone (GH)-releasing hormone (GRH) and GRH receptor in various rat tissues. Endocrinology 1995;136:4147–50.