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Widespread Organ Expression of the Rat Proenkephalin Gene during Early Postnatal Development David Kew and Daniel L. Kilpatrick Neurobiology Group Worcester Foundation for Experimental Biology Shrewsbury, Massachussetts 01545 RESULTS The opioid peptides have been implicated as potential regulators of cell development in nervous and reproductive tissues. A survey of proenkephalin gene expression during rat development showed that the mRNA for this opioid precursor is present at substantial concentrations in several developing tissues (kidney, liver, skin, skeletal muscle, and lung) that have essentially undetectable levels in adults. In neonatal rats, skeletal muscle has greater concentrations of this transcript than brain. Polysomal analysis further demonstrated that proenkephalin mRNA is actively translated in skeletal muscle from newborn rats. These results raise the possibility that proenkephalin and its products perform a general regulatory role in cell proliferation or differentiation. (Molecular Endocrinology 4: 337-340, 1990) Proenkephalin mRNA in Various Tissues of Immature and Adult Rats A comparison of the relative abundances of proenkephalin mRNA was initially made between tissues from 14-day-old and adult (90- to 115-day-old) rats. In both age groups the concentrations of this transcript were relatively high in brain and heart, but were below the limits of detection in kidney, liver, and spleen (Fig. 1; see also Fig. 2). In contrast, proenkephalin mRNA was readily detectable in skeletal muscle and lung from 14day-old rats, but was either markedly reduced in concentration (lung) or undetectable (muscle) in the adult organs. The size of this transcript was the same as that observed in brain and other adult somatic tissues [1450 nucleotides (8)]. Speculating that expression of the proenkephalin gene may be correlated with rapid growth or differentiation, we then assayed the organs of neonatal rats (1-2 days of age). The results demonstrated that the proenkephalin gene was more widely expressed than at the older ages (Fig. 2). In addition to neonatal brain, heart, and lung, proenkephalin mRNA was also observed in newborn skeletal muscle, liver, kidney, skin, and intestine, tissues that have negligible or undetectable transcript levels in the adult (see Fig. 1). The only organ from neonatal rat pups examined in this survey that did not contain detectable amounts of proenkephalin mRNA was the spleen (Fig. 2). It should be noted that the abundance of proenkephalin mRNA observed in developing nonneural tissues is quite substantial. For example, expression in neonatal skeletal muscle is greater than that observed in neonatal brain (Fig. 2). Transcript abundance in muscle shows a steady several-fold decline during the first 2 weeks after birth and falls below the limits of detection in the adult (Fig. 3). In contrast, both brain and heart exhibited the opposite pattern of developmental expression, with proenkephalin mRNA levels being substantially higher in the adult tissues (Fig. 2). INTRODUCTION Peptide growth factors play a major role in the control of cell development. Recent studies have suggested that peptides typically associated with neural and/or endocrine functions also have growth-promoting effects and may be involved in tumorigenesis (1, 2). Included in this group are opioid peptides such as /3-endorphin and the enkephalins. /S-Endorphin has modulatory effects on the proliferation of lymphocytes and cell lines derived from small cell carcinoma of the lung (2, 3). Opioid peptides derived from proenkephalin have been implicated as regulators in the early development of neuronal and glial cells (4,5). Further evidence suggests that proenkephalin and its products may play a more general role in tissue development. For example, the proenkephalin gene is expressed by testicular somatic cells during postnatal development (6) and by developing spermatogenic cells in the adult rat (7). We, therefore, sought to determine the pattern of developmental expression for the proenkephalin gene in different rat organs and tissues. Our results are consistent with the hypothesis that proenkephalin-derived peptides are involved in cell development in a variety of tissues. Polysome Distribution of Proenkephalin mRNA in Developing Muscle 0888-8809/90/0337-0340S02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society An important question arising from the foregoing data is whether proenkephalin mRNA present exclusively 337 Vol 4 No. 2 MOL ENDO-1990 338 during development is functional, i.e. is translated into proenkephalin. This was addressed by examining the polysome distribution of this transcript in neonatal rat skeletal muscle, which has relatively high concentrations of proenkephalin mRNA at this age. This approach was employed instead of peptide quantitation since several tissues that contain and actively translate proenkephalin mRNA do not exhibit significant concentrations of proenkephalin products, apparently due to a rapid rate of peptide secretion or turnover (6, 9). Proenkephalin mRNA was localized to the polysomal fractions in gradients prepared from neonatal skeletal muscle (Fig. 4). This polysome profile is comparable to that observed for other tissues previously shown to efficiently translate proenkephalin mRNA, such as brain (6). Proenkephalin transcripts were released from polysomes in the presence of EDTA (Fig. 4), indicative of specific polysomal association. This transcript is, therefore, actively translated during postnatal muscle development. Although not specifically examined here, it is likely that proenkephalin mRNA is also efficiently translated in other developing organs in which it is present. -I8S Fig. 2. Comparison of Proenkephalin mRNA in Neonatal (neo.) and Adult (ad.) Rat Tissues Each lane contained 20 ng poly(A)+ RNA. A, Neonatal tissues and two adult tissue controls. B, Direct comparison of neonatal and adult brain RNAs. This is a shorter exposure than in A. DISCUSSION The results reported here indicate that the opioid peptide precursor proenkephalin is produced by a variety of rat tissues during early postnatal development. This group includes tissues derived from embryonic endoderm (liver and lung), mesoderm (skeletal muscle, heart, and kidney), and ectoderm (brain). Skin has both ectodermal (epidermis) and mesodermal components (dermis), and intestine has both endodermal and mesodermal origins, the relative contributions of which were not determined here. In most cases, the level of proen- -I8S Fig. 1. RNA Gel-Blot Analysis of Proenkephalin mRNA in Several Adult and Juvenile (14-Day-Old) Rat Tissues Each tissue is represented by 30 ^g poly(A)+ RNA. Samples were prepared and analyzed as described in Materials and Methods. A, Fourteen-day-old tissues. B, Adult tissues. t- -I8S Fig. 3. Age-Dependent Changes in the Abundance of Proenkephalin mRNA in Rat Skeletal Muscle Each lane contained 20 ng poly(A)+ RNA. RNA gel-blot analysis was performed as outlined in Materials and Methods. kephalin gene expression is markedly reduced or undetectable in the adult rat. Two exceptions to this pattern are brain and heart, which exhibit higher proenkephalin mRNA abundance in the adult. This probably reflects mRNA production by cells programed to express the proenkephalin gene at relatively high levels in the mature animal. It, therefore, appears that in addition to its production by certain fully differentiated tissues (e.g. brain, adrenal medulla, heart, and reproductive organs), proenkephalin expression is also associated with cell proliferation or differentiation. The absence of proenkephalin mRNA in postnatal rat spleen may indicate that this association is not general, although perhaps developmental expression of proenkephalin in this organ is limited to the fetal period, which was not examined here. More extensive analysis, using in situ hybridization, will be necessary to further identify and characterize the proenkephalin-expressing cells and their fates during the development of different tissues. 339 Developmental Expression of Proenkephalin HKMG -18 S HKE I top -18 S 8 9 10 II 12 bottom Fig. 4. Polysome Profile of Proenkephalin mRNA in Neonatal Rat Skeletal Muscle Polysomes are intact in sucrose gradients containing HKMG buffer and are dissociated in HKE gradients (containing EDTA in place of Mg2+). The preparation and analysis of postmitochondrial supernates are described in Materials and Methods. The direction of sedimentation is from left to right as shown. Based on the absorbance profile at 260 nm (not shown), polysomes were localized to fractions 7-12, and the postpolysomal region was found in fractions 1-3. The recent demonstration of proenkephalin expression by developing astrocytes and neurons (4, 5, 10) suggests that the peptide products derived from this precursor may regulate cell differentiation or proliferation within the brain. Growth-promoting effects of enkephalins and other opioid peptides on cultured neurons and glial cells have been reported (11,12). The much wider tissue distribution of proenkephalin gene expression during early postnatal development and its subsequent decline with age shown here are suggestive of a more general association with cell growth. Proenkephalin gene expression in immature testes and in developing male germ cells is consistent with this notion. As pointed out above, the abundance of proenkephalin mRNA in certain nonneural tissues (e.g. skeletal muscle) is even greater than that observed in brain at early stages of development. Bombesin-like peptides are selectively produced in lung during development and have been shown to stimulate cell proliferation (2). Other neuropeptides that have been implicated as growth regulators include vasopressin, gastrin, the neuromedins (substance-P and substance-K), and /3-endorphin (2). Proenkephalin products, therefore, may be part of a class of peptides with both growth-related activities during development as well as neuro/endocrine functions in fully differentiated tissues. This apparent duality of peptide function may have evolved long ago, as indicated by the neurotransmitter and mitogenic/differentiative activities exhibited by the head activator peptide in the coelenterate Hydra (13). The work reported here generally supports the conclusions of previous immunohistochemical analyses of enkephalin-like immunoreactivity in developing rat tissues, with at least one exception. Zagon et al. (14) observed substantial immunoreactivity in immature rat spleen, while we found no proenkephalin mRNA. This underlines the necessity for biochemical characterization of detected immunoreactivity, which was not performed in these earlier studies. The relationship of this enkephalin-like material to authentic enkephalin or en- kephalin-containing peptides derived from proenkephalin, therefore, remains uncertain. An understanding of the possible role of proenkephalin in tissue development will ultimately follow determination of the specific peptide products synthesized and secreted by developing cells. Another intriguing question raised by these studies concerns the mechanism(s) responsible for the decline in proenkephalin mRNA abundance with age. One possibility is that this reflects development-dependent repression of proenkephalin gene transcription, as described for a-fetoprotein (15). Posttranscriptional processes (e.g. mRNA destabilization) also may contribute. Gene transfer will be a valuable approach in examining these possibilities. MATERIALS AND METHODS Animals and Tissues Adult and newborn CD-1 rats (Charles River Laboratories, Wilmington, MA) were killed by CO2 inhalation, and tissues were either extracted immediately or frozen on dry ice and stored at - 8 0 C for later analysis. The skeletal muscle used was from the thigh, shoulder, and upper forelimb. Tissue Comparisons Fresh or frozen tissues were weighed and homogenized in approximately 10 vol guanidine thiocyanate buffer with a Tekmar homogenizer, and the RNA was collected by centrifuging through CsCI buffer (16). Poly(A)+ RNA was selected with oligo-dT (Pharmacia, Piscataway, NJ). Equal amounts of poly(A)+ RNA were separated on formaldehyde-agarose gels, and blotted to Gene-Screen Plus membranes (New England Nuclear, Boston, MA). Blots were probed with cDNA to rat proenkephalin [pRPE-1 (165-600)] (8) labeled by random priming with [32P]dCTP. Blots and probe were incubated overnight at 42 C, with final washes at 56 C in 15 mM NaCI-1.5 ITIM sodium citrate-0.1% sodium dodecyl sulfate (17,18). Analyses were performed on at least two different RNA preparations in each case. Vol 4 No. 2 MOL ENDO-1990 340 Polysome Analysis The protocol was based on that of Kleene et al. (19). Fresh newborn skeletal muscle was homogenized in a motorized glass/Teflon homogenizer at 4 C in 4 vol HKMG buffer (20 miui HEPES-100 mM KCI-20 miui MgCI2-10 mM EGTA, pH 7.6) containing 0.5% (wt/vol) Triton X-100-3 ITIM mercaptoethanol200 U/ml RNAsin (Promega, Madison, Wl). Tissue for the nonspecificity control was homogenized in HK buffer (20 mM HEPES-100 mM KCI, pH 7.6), also containing Triton X-100, mercaptoethanol, and RNAsin. Homogenates were centrifuged to pellet nuclei and debris, then 0.5 M EDTA (pH 7.6) was added to the HK sample to a final concentration of 20 mM (HKE buffer) to dissociate the polysomes. The supemates were overlaid onto linear gradients of 10-40% (wt/wt) sucrose with 60% sucrose cushions, made up in HKE or HKMG buffer as appropriate. Gradients were spun in a Beckman SW28 rotor (Beckman, Palo Alto, CA) for 2.5 h at 28,000 rpm at 4 C, and decelerated without the brake. Gradient fractions were diluted with an equal volume of 0.4 M sodium acetate (pH 5.5), mixed with 50 (ig yeast tRNA and 20 ng glycogen carrier, and precipitated at - 2 0 C overnight with isopropanol. RNA was extracted and purified further by the method of Chomczynski and Sacchi (20). Fractions were dissolved in guanidine thiocyanate buffer, extracted with phenol-chloroform-isoamyl alcohol, and again precipitated with isopropanol. Precipitated fractions were dissolved in 0.5% sodium dodecyl sulfate and reprecipitated with ethanol. Equivalent percentages of each fraction were run on a gel, blotted, and probed for proenkephalin mRNA as described above. Note Added in Proof Two additional reports describing the developmental expression of proenkaphalin have recently appeared: Keshet E, Polakiewicz RD, Itin A, Orney A, Rosen H 1989 Proenkephalin A is expressed in mesodermal lineages during organogenesis. EMBO J 8:2917-2923; Springhom JP, Claycomb WC 1989 Proenkephalin mRNA expression in developing rat heart and in cultured ventricular cardiac muscle cells. Biochem J 258:73-78. Acknowledgments We thank Dr. Steven Zinn and Mr. Carl Schell for their help. Received October 9, 1989. Revision received November 14,1989. Accepted November 16,1989. Address requests for reprints to: Dr. Daniel L. Kilpatrick, Worcester Foundation for Experimental Biology, Neurobiology Group, Shrewsbury, Massachussetts 01545. This work was supported by NIH Grants DK-35855 and DK36486 (to D.L.K.) and grants to the Worcester Foundation from the Andrew W. Mellon Foundation and the Edward John Noble Foundation. REFERENCES 1. 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Muffly KE, Jin DF, Okulicz WC, Kilpatrick DL 1988 Gonadal steroids regulate proenkephalin gene expression in a tissue-specific manner within the female reproductive system. Mol Endocrinol 2:979-985 18. Kilpatrick DL, Rosenthal JL 1986 The proenkephalin gene is widely expressed within the male and female reproductive systems of the rat and hamster. Endocrinology 119:370-374 19. Kleene KC, Distel RJ, Hecht NB 1984 Translational regulation and deadenylation of a protamine mRNA during spermiogenesis in the mouse. Dev Biol 105:71-79 20. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-159