Differential coexpression of genes encoding prothyrotropin-releasing hormone (pro-TRH) and prohormone convertases (PC1 and PC2) in rat brain neurons: implications for differential processing of pro-TRH

0013.7227/96/$03.00/O Endocnnolagy CopynghL 0 1996 by The Endocrine Vol. 137, No. 4 Prlntrd ,n U.S.A. Society Differential Coexpression Prothyrotropin-Releasing Prohormone Convertases Neurons: Implications for Pro-TRH of Genes Encoding Hormone (Pro-TRH) and (PC1 and PC2) in Rat Brain Differential Processing of LE-PING AND Y. PENG LOH PU, WU MA, JEFFERY L. BARKER, Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institutes of Child Health and Human Development (L.P.P., Y.P.L.), and the Laboratory of Neurophysiology, National Institutes of Neurological Disorders and Stroke (W.M, J.L.B.), National Institutes of Health, Bethesda, Maryland 20892 ence of pro-TRH mRNA with PC2 mRNA, but not PC1 mRNA. Interestingly, pro-TRH was expressed in the thalamic reticular nucleus, but neither PC1 nor PC2 was detectable in this region. Cellular colocalization studies using double in situ hybridization histochemistry showed the presence of PC2 mRNA in the pro-TRH neurons of the olfactory glomerular layer and basal lateral hypothalamus, and PC1 mRNA in the pro-TRH neurons in the paraventricular nucleus. These results suggest that PC1 and PC2 are enzyme candidates for the processingofpro-TRH in uiuo. Moreover, the differential distribution of PC1 and PC2 mRNAs with pro-TRH mRNA may be responsible for the differential processing of this prohormone in the central nervous system. The absence of PC1 and PC2 mRNAs in certain TRH neurons raises the possibility that prohormone convertases other than PC1 and PC2 may be involved in the processing of brain pro-TRH. (Endocrinology 137: 12331241, 1996) ABSTRACT Pro-TRH is cleaved at paired basic residues to yield five copies of TRH and cryptic peptides. Recent studies have shown that the prohormone convertases, PC1 and PC2, can process pro-TRH correctly. To determine whether these two enzymes could play a role in pro-TRH processing in uiuo, the regional and cellular colocalization of pro-TRH messenger RNA (mRNA) with the mRNAs encoding the prohormone convertases PC1 and PC2 was examined in rat brain, using in situ hybridization histochemistry. Differential regional distribution of pro-TRH mRNA with PC1 and/or PC2 mRNA was found in several brain regions. For example, in the olfactory regions, there was coexpression of pro-TRH mRNA in the glomerular layer with PC2 mRNA, but not PC1 mRNA, whereas in the tenia tecta, coexpression of pro-TRH and PC1 mRNAs was evident, but PC2 mRNA was absent. Pro-TRH mRNA in the paraventricular nucleus was coexpressed with both PC1 and PC2 mRNAs, whereas the basal lateral hypothalamus showed coexist- D IFFERENTIAL posttranslational processing of multivalent peptide precursors in neurons and endocrine cells is a principle mechanism regulating neuropeptide and hormone biosynthesis and, hence, neuroendocrine function (l-3). Recently, a mammalian Kex2/subtilisinlike family of proteases has been cloned and implicated as processing enzymes for various proproteins (4-7). These include furin (5, 8), prohormone convertase 1 (Xl; also known as PC3) (6, 9), PC2 (2), PC4 (10, ll), PACE4 (12), PC5 (13), and PC6 (14). Analysis of the tissue distribution of these convertases showed a unique pattern for each enzyme. Of these, only PC1 and PC2 are mainly confined to the neuroendocrine tissues, including the brain (6, 1517). Thus, PC1 enzymes involved the neuroendocrine system (CNS). and PC2 are the most likely In the present study, we have focussed on determining whether PC1 and PC2 are potential enzymes for cleaving the TRH precursor (pro-TRH) in viuo and in a differential manner in brain. Based on the deduced amino acid sequence of rat pro-TRH, processing at all paired basic residues of this precursor is expected to produce five copies of TRH along with seven other non-TRH cryptic peptides (Fig. 1). However, previous studies have shown the presence of TRH, non-TRH-containing cryptic peptides, and/or extended forms of TRH-related peptides in various rat brain regions (18-20), indicating differential processing of pro-TRH in rat CNS. In zlifro studies have demonstrated that PC1 and PC2 can differentially process proTRH (21, 22). In this study, we show differential distribution and cellular colocalization of PC1 and/or PC2 messenger RNA (mRNA) with pro-TRH mRNA in various regions of adult rat brain using single/double labeling in situ hybridization histochemistry. The data provide evidence suggesting that PC1 and PC2 are enzyme candidates for processing pro-TRH in zlivo and may act separately, in combination, or with other processing enzymes to differentially process pro-TRH in a cell- and/or region-specific manner in the mammalian CNS. candidate in the activation of proneuropeptides in system, including the central nervous Received July 12, 1995. Address all correspondence and requests for reprints to: Dr. Y. I’. Loh, Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institutes of Child Health and Human Development, National Institutes of Health, Building 49, Room 5A38, Bethesda, Maryland 20892. 1233 1234 COEXPRESSION OF PRO-TRH, PCl, AND PC2 mRNAs IN Endo. 1996 Vol 137 . No 4 BRAIN 255 1 FIG. 1. Schematic representation of the structure of rat pro-TRH. The positions of the TRH progenitor sequences, Gln-His-Pro-Gly, which are flanked by pairs of basic amino acid residues, are shown in solid bars. The hatched bar represents the putative signal peptide sequence. There is also an additional pair of basic amino residues at the N-terminal of the rat pro-TRH molecule that may be a potential cleavage site. Materials Animals and Methods and tissue preparation Adult male Sprague-Dawley rats, weighing 220-250 g, were obtained from a commercial source (Taconic Farms, Germantown, NY). The animals were maintained in a temperature-controlled room (20 t 0.5 Cl under a 12-h light, 12-h dark schedule, with free access to drinking water and commercial food pellets. Animals were anesthetized with chloral hydrate (400 pg/kg, ip), and the brain was carefully dissected out and rapidly frozen on dry ice. Serial sections (12 ym) were cut throughout the whole brain in sagittal or coronal planes on a cryostat, thaw-mounted onto gelatin-coated microscope slides, and stored at -70 C until further processing. Preparation of complementary RNA (cRNA) probes PCZ. pBS.rPCl-491EX (a gift from Drs. Mains and Eipper, Johns Hopkins University, Baltimore, MD) is the plasmid BS.11 SK(-) with a 491-bp insert encoding an N-terminal portion of rat PC1 (l-491) (23,24). This probe has little homology with rat PC2 and shows no cross-hybridization (24). To synthesize a 35S-labeled antisense cRNA probe for rat PC1 mRNA, pBS.rPCl-491EX was linearized with EcoRI and in vitro transcribed in the presence of T3 RN’A polymerase and 35S-labeled UTP (New England Nuclear-DuPont, Boston, MA). A sense cRNA probe was transcribed with T7 RNA polymerase after linearizing with Ba (...truncated)