Expression of Protein Kinase C Genes during Ontogenic Development ...

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Oct 20, 1988 - 263:4523-4526. 10. Housey, G. M., C. A. O'Brian, M. D. Johnson, P. Kirschmeier, and I. B. Weinstein. 1987. Isolation of cDNA clones encoding.
MOLECULAR AND CELLULAR BIOLOGY, May 1989, p. 2284-2288 0270-7306/89/052284-05$02.00/0 Copyright X 1989, American Society for Microbiology

Vol. 9, No. 5

Expression of Protein Kinase C Genes during Ontogenic Development of the Central Nervous System N. M. SPOSI,1* L. BOTTERO,1 G. COSSU,2 G. RUSSO,3 U. TESTA,' AND C. PESCHLE' Department of Hematology & Oncology, Istituto Superiore di Sanita, Viale Regina Elena, 299, 00161 Rome'; Institute of Histology and Embryology, University La Sapienza, Via Scarpa, 141, 00161 Rome2; and Division of Obsteirics and Gynecology, Ospedale Civile di Avellino, 83100 Avellino3 Italy Received 20 October 1988/Accepted 14 February 1989

We have analyzed the RNA expression of three protein kinase C (PKC) genes (a, f, and y) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC a and ji genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC y gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC -y is induced in the peri- and postnatal phases. These results suggest that expression of PKC a, 3, and y genes possibly mediates the development of central neuronal functions, and expression of PKC -y in particular may be involved in the development of peri- and postnatal functions.

Protein kinase C (PKC) is a Ca2+- and phospholipiddependent serine-threonine protein kinase, first isolated from rat brain by Nishizuka (13, 23). It is ubiquitously distributed in tissues and organs, with the highest level of activity observed in brain cells (17, 20). Numerous hormones, growth factors, and neurotransmitters, acting at the level of membrane receptors, stimulate the phosphodiesterasic hydrolysis of phosphatidylinositol 4,5biphosphate. This generates two second messengers, inositol 1,4,5-triphosphate and diacylglycerol (3, 14), which binds to and stimulates PKC activity (24). This action is mimicked by phorbol esters (1). It is now generally accepted that the PKC enzymatic complex plays a crucial role in the control of a wide variety of physiological processes such as cell differentiation, tumor promotion, and membrane protein function (for a review, see reference 25). Recently, cDNA clones encoding distinct forms of PKC have been isolated, thus indicating that PKC is a multigene family (7, 10, 15, 16, 18, 27-29, 32-34). Four distinct PKC isozymes isolated from a variety cDNA libraries (7, 10, 15, 16, 18, 27-29, 32-34) were designated a, pi, P2, and y. The correspondence between different cDNA clones and PKC isozymes, although suggested (26), has not been unequivocally established yet. More recently, at least three other PKC subspecies (8, r, and ;) have been isolated from a rat brain cDNA library by using a mixture of a, P2, and y cDNA probes under low-stringency conditions (30, 31). These three subspecies have a common structure which is closely related to, but clearly distinct from, that of the other four subspe-

expression of different PKC isoforms in the central nervous system (CNS) suggests that these enzymes are involved in a variety of neuronal functions (cf. references 4, 22, 26, and 38). The expression of PKC genes has not yet been explored in the early stages of ontogeny. We have analyzed the RNA expression of three PKC isoforms (a, P, -y) during the ontogenic development of human and murine CNSs, i.e., in embryonic-fetal, perinatal, and adult stages of life. We used specific PKC a (34), p (hybridizing to both P1 and P2 RNA, [32]), and -y (7) human cDNA probes to analyze by Northern (RNA) blotting the poly(A)+ RNA extracted from the brain cells of human embryos and fetuses, which had been obtained virtually intact from legal curettage abortions (35). Fertilization ages of 6- to 8-week-old embryos were established by morphological staging according to multiple criteria (21); 9-week-old fetuses were staged on the basis of standard age and crown-rump length plots (21). The integrity of the examined embryos and fetuses allowed a dating error of as little as ±2 days (21). Tissues of equivalent ages were pooled. We then extended our studies to total RNA from normal human adult brain cells obtained by biopsy during surgical excision of a brain tumor. Total RNA was extracted by the guanidinium thiocyanate technique (5), and poly(A)+ was selected by one passage on oligo(dT) cellulose columns (2). A 10-p.g sample of total RNA or 2 to 3 ,ug of poly(A)+ RNA were run on 1.0% agarose-formaldehyde gels (19), transferred to nylon membranes (Hybond N; Amersham Corp., Arlington Heights, Ill.) by Northern capillary blotting (37), and hybridized to 107 cpm of a cDNA probe labeled by nick translation to a specific activity of 3 x 108 to 8 x 108 dpm/,lg. Prehybridization and hybridization were carried out as described (37). After washing under stringent conditions (15 mM NaCl, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 65°C),

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Despite their closely related structures, the different PKC isoforms exhibit considerable tissue specificity. This suggests that each of them may mediate specific functions in different tissues (4, 10, 16, 27). In particular, the abundant *

Corresponding author. 2284

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the blots were exposed for 1 to 3 days at -70'C to Kodak XR-5 film in an X-Omat intensifying screen box. After the first hybridization, the PKC a probe was removed by boiling in 0.1% sodium dodecyl sulfate-buffered solution, and the filter was rehybridized to the PKC 13 probe. Dehybridization was controlled by overnight exposure at -70'C to Kodak XR-5 film in an X-Omat intensifying screen cassette. The same procedure was used for rehybridization to PKC -y probe. Rehybridization to a chicken 13-actin probe (6) was carried out to normalize RNA levels. Appropriate autoradiograms were scanned by densitometry, and the expression of mRNAs was quantitatively evaluated. Northern blot analysis of poly(A)' RNA from 6- to 9-week-old brain cells indicated that the PKC a gene is expressed in a single major transcript of 9.5 kilobases (kb). Experiments with total RNA from adult brain showed a similar pattern (Fig. 1). Hybridization of poly(A)' RNA from 6- to 9-week-old brain cells with a PKC 13 cDNA probe showed two maj'or transcripts of 8.9 and 4.2 kb, respectively, and a minor band of 3.4 kb. Analysis of adult brain showed the same transcripts (Fig. 1 and 2). It is noteworthy that the 3.4-kb PKC 13 RNA was undetectable in 6-week-old brain cells. The multiple mRNA transcripts may derive from alternative initiation or termination sites of transcription with or without differential posttranscriptional processing of primary transcripts (34). The remote possibility exists that these different transcripts are encoded by closely related but distinct genes. In contrast to the findings observed for PKC a and 13 genes, PKC -y mRNA was not detected in 6- to 9-week-old brain cells (Fig. 1). Conversely, expression of PKC -y RNA was abundant in the adult brain as a single transcript of 3.8 kb (Fig. 1). In conclusion, these observations on human brain essentially indicate the following. (i) The expression of PKC a gene, barely detected in 6-week-old cells, undergoes a gradual increase until cells are 9 weeks old and reaches its peak in adult cells, i.e., the mRNA level is -10-fold higher in adult brain than in 9-week-old fetal brain. (ii) Both the 8.9-kb and 4.2-kb PKC 13 transcripts show a low, comparable abundance in 6- to 9-week-old cells which undergoes a --30-fold increase in the adult stage. The 3.4-kb PKC 13 transcript, apparently absent and hardly detectable in 6week-old and 9-week-old brain, respectively, reaches a peak level of expression in the adult period, comparable to that observed for 8.9- and 4.2-kb transcripts. (iii) The PKC -y gene, although apparently not transcribed during embryonic and early fetal development, is abundantly expressed in the adult period. We extended our analysis to poly(A)' RNA from different regions of 6- to 8-week-old embryonic CNS. The CNS was dissected at the level of the pontine and cervical flexures, thus separating fore- and midbrain from the medulla oblongata and the spinal cord, respectively. The pattern in medulla oblongata and spinal cord (Fig. 3; results not shown) was virtually identical to that observed for the brain; PKC a and 13 genes were expressed at progressively increasing levels in 6- to 8-week-old cells whereas the expression of PKC -y gene was undetectable. Furthermore, higher levels of PKC a and 13 RNA were observed in spinal cord than in

medulla oblongata.

A more detailed analysis of the expression of PKC genes during mammalian development was performed on poly(A)' RNA from the brain cells of murine embryos at 10, 13, and 16 days postcoitum and from newborn or adult mice. Em-

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FIG. 1. Northern blot analysis of PKC a, 13, and -y transcripts in total (lane c) or poly(A)' (lanes a and b) RNA from normal human brain at various stages of development. Lanes a through c represent 6-week-old, 9-week-old, and adult brain, respectively. Embryonicfetal specimens of corresponding ages were pooled. The probes were cDNA inserts hPKC a (1,294-base-pair EcoRI-EcoRI) (A), hPKC 13 (1,702-base-pair EcoRI-EcoRI) (B), and hPKC -y (1,397base-pair EcoRI-EcoRI) (C). Sequence homologies between corresponding regions of the PKC a, 13, and -y cDNAs were as follows: a and 13, 74%; at and -y, 71%; 13 and a, 73%; 13 and -y, 68%; -y and a, 68%; -y and 13, 66%. To exclude cross-hybridizations, we employed high-stringency conditions (see text). The faint 9-kb band observed after rehybridization with the PKC -y probe is seemingly due to a residual 9-kb PKC 13 band. The filter was rehybridized to a chicken 13-actin probe (D) (38) for normalization. Numbers on the right are sizes in kilobases (RNA markers were obtained from Bethesda Research Laboratories). Arrowheads indicate positions of 28S rRNA.

bryos were isolated in phosphate-buffered saline. The brain or brain vesicles were dissected under the microscope. Northern blot analysis indicated that the PKC a gene is gradually activated during embryonic life and expressed in a major transcript of 9.5 kb, as well as in faint 3.8- and 4.3-kb bands (Fig. 4). Hybridization with the PKC 13 probe showed a similar ontogenetic pattern with two major transcripts of 9.5 and 3.8 kb, respectively (Fig. 4). Quantitative analysis of RNA levels showed that PKC a gene expression is -10-fold more elevated in adult brain than in 16-day-old embryonic or newborn brain. The expression of the 9.5- and 3.8-kb PKC 13 RNA shows a twofold increase from embryonic to neonatal life, with a further -10-fold rise in the adult brain. Hybridization to a PKC -y cDNA probe showed a major transcript of 4.1 kb, which was abundant in adult brain but

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