Expression of gap junction genes in astrocytes and C6 glioma cells

Neuroscience Letters, I26 (199 1) 33-36 0 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$03.50 ADONIS0304394091002088

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NSL 07727

Expression of gap junction genes in astrocytes and C6 glioma cells Christian C.G. Naus’, John F. Bechberger’, Stan Caveney2 and John X. Wilson3 Departments of ‘Anatomy, 2Zoology and 3Physiology, The University of Western Ontario, London, Ont. (Canada)

(Received 25 June 1990; Revised version received 4 January 1991; Accepted 6 February 1991) Key words:

Gap junction; Connexin43; Astrocyte; Glioma

The expression of the gap junction genes coding for the liver-type connexin32 and the heart-type connexin43 was examined in primary cultures of astrocytes and in cultures of C6 glioma cells. In both cell types, only connexin43 mRNA was detectable. However, the level of this mRNA was greatly reduced in C6 glioma cells compared to astrocytes. This was consistent with the further observation that astrocytes in primary culture were extensively dye-coupled, whereas such coupling was very restricted in cultures of C6 glioma cells. Connexin43 was immunocytochemically localized in astrocytes, but was not readily detected in C6 cells.

Gap junction proteins are encoded by a family of genes, the most studied being the gap junction proteins initially isolated from liver, connexin32 and connexin26, and heart, connexin43 [3, 13, 20, 221. Reports from our laboratory, as well as others, have shown that the mRNAs coding some of these gap junction proteins are expressed in human and rodent brain [17, 18, 221. Recently, in situ studies have suggested a cell specificity in the expression of these gap junction genes, with connexin32 mRNA [ 191and connexin32-like immunoreactivity (LI) [5, 271 being localized to oligodendrocytes and some neurons, while connexin43 mRNA [19] and connexin43-LI [28] has been associated with astrocytes and endothelial cells. Another approach to resolve specificity would be to prepare cultures of various neural cell types and assay for different connexin mRNAs and functional gap junctions. Reports identifying specific gap junction proteins or mRNAs present in astrocytes are inconsistent. While a 27-kDa protein cross-reacting with an antibody to livertype gap junctions has been demonstrated in astrocyte cultures [6], other investigators report the expression of the heart-type gap junction, connexin43, in astrocytes in situ [5, 7, 281. Here, we show that primary cultures of astrocytes and C6 glioma cells express only the connexin43 mRNA, and that the level of this mRNA is dramatically reduced in the glioma cells. Glial cultures were prepared as previously described by Hertz et al. [ll]. Briefly, neopallium of newborn Correspondence: C.C.G. Naus, Department of Anatomy, University of Western Ontario, London, Ont., Canada N6A X1.

The

Sprague-Dawley rats was disrupted by trituration and vortex-mixing in modified Eagle’s minimum essential medium (MEM) (G&co) with 20% horse serum. Cells were passed twice through sterile nylon sieves (10 pm) prior to seeding in petri dishes or on sterile coverslips and incubated at 37°C in 95% atmospheric air/S% CO2 with 90% relative humidity. Serum concentration was reduced to 10% after 1 week, and cultures reached confluency after 2 weeks. They were then grown with or without 0.25 mM dibutyryl CAMP, and harvested 2 weeks later. Previous work from our laboratory has indicated that greater than 80% of these cells in primary astrocyte cultures are immunoreactive for glial fibrillary acidic protein (GFAP) [26]. C6 glioma cells were cultured as previously described [ 11.The C6 astrocytoma cell line [2] (American Type Tissue Culture) was established in monolayer culture in MEM supplemented with 5% fetal calf serum, penicillin G (100 U/ml) and streptomycin (100 pg/ml). Total RNA was extracted and fractionated for Northern blot analysis of GFAP mRNA and connexin mRNAs. Using methods previously employed in our laboratory [17], RNA was isolated from cultures and fractionated by electrophoresis on 1.5% agarose gels in the presence of 1 M formaldehyde and transferred to nitrocellulose. The cDNAs for rat connexin32 [22] and connexin43 [3] (kindly provided by D.L. Paul, Harvard University) were radiolabeled with 32P by random priming and simultaneously hybridized to the immobilized RNA. Hybridization was performed at 42°C in 50% formamide, 5 x Denhardt’s, 5 x PIPES, 0.2% SDS, and posthybridization washes were done at room tempera-

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ture in 2 x standard saline citrate (SSC), 0.2% SDS, followed by 0.5 x SSC, 0.2% SDS at 37°C and 0.15 x SSC at 65°C. The same blot was later hybridized with a radiolabeled cDNA for GFAP. To control for lane loading and possible variability in RNA transfer, the same membranes were hybridized with a radiolabeled cDNA for 1% rRNA. In all cases, radiolabelled Northern blots were apposed to X-ray film and the resultant images scanned to obtain densitometric values (Ultrascan XL, LKB). To test for functional intercellular coupling via gap junctions, single cells were injected with the fluorescent dye, 6-carboxyfluorescein (10 mM in distilled water, pH 7.0; Eastman Kodak), using a continuous train of hyperpolarizing current pulses of 2-6 nA (200 ms duration, I/ s). The quality and stability of cell penetration were monitored by simultaneous membrane potential recordings and dye injections were recorded as previously described [23]. To localize connexin43 at the cellular level, cultures of astrocytes were fixed in 95% ethanol/5% glacial acetic acid and incubated in a mixture of affinity purified rabbit polyclonal anti-connexin43 (0.089 mg/ml) (gift from B.J. Nicholson; diluted 1:lOO) and mouse monoclonal antiGFAP (Boehringer Mannheim; diluted 1:4). This was followed by incubation in fluorescein-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat antimouse IgG (Vector Labs.; diluted 1:50).

Using RNA analysis, we have previously found that both connexin43 and connexin32 mRNA are relatively abundant in adult brain [17]. This is evident in Fig. IA (lane I), where two bands of hybridization can be seen; connexin43 at approximately 3 kb, and connexin32 at approximately 1.6 kb. In contrast, primary astrocytes expressed only one of these gap junction genes, namely connexin43 (Fig. IA, lane 2). Compared to RNA isolated from adult hindbrain, the level of connexin43 was enriched in astrocyte cultures. Treatment of these primary cultures with dbcAMP changed their morphology from polygonal to stellate and increased the level of GFAP mRNA (Fig. 1B), as previously reported [24], but did not alter the level of connexin43 mRNA (Fig. IA, lane 3). The cultures of C6 cells also contained only connexin43 mRNA (Fig. IA, lane 4). Of particular significance is the observation that the level of connexin43 mRNA was dramatically decreased in these glioma cells. Densitometric analysis indicated that the level of this mRNA was approximately IO-fold higher in the astrocyte cultures compared to the C6 cells. In addition, the level of GFAP mRNA was also reduced in the C6 cells compared to either polygonal or stellate astroglial cells (Fig. 1B, lane 4). Dye-coupling studies indicated that this lower level of connexin43 mRNA in the C6 cells, relative to the primary astrocytes, was reflected in a lower incidence of intercellular coupling via gap junctions (Fig. 2). In all 8 trials with cultures of polygonal and stellate astrocytes, there was rapid diffusion of dye from the single injected cell (Fig. 2A) to adjacent astrocytes (Fig. 2B,C). However, in 23 intracellular injections of C6 glioma cells, only 2

cx32 . I

A

Fig.

I. Northern

isolated

blot analysis

from adult

rat brain

of connexin43

(lane 2) and stellate (lane 3) astrocytes, (lane 4). The blot was initially hybridized connexin43

and connexin32

and connexin32

(lane 1), primary

cultures

and cultures

of C6 glioma cells

with radiolabeled

(A), and later rehybridized for GFAP (B).

in RNA

of polygonal cDNAs

for

with the cDNA

Fig. 2. Examination

of dye-coupling

between primary

astrocytes

(A-C)

and between C6 cells (D-F). Micrographs were taken at the time of impalement (A,D). and 90 s (B,E) and 180 s (C,F) later. Bar = 50 pm.

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Fig. 3. Immunocytochemical

staining

immunoreactive

for connexin43 astrocytes,

(A) and GFAP

similar staining

(B) in astrocytes.

for gap junctions

cases displayed a very low level of intercellular transfer of dye and the remainder showed no transfer (Fig. 2C,D,E). Since some of the cells in the astrocyte cultures are not immunoreactive for GFAP [26], the connexin43 mRNA detected in these cultures may be non-astrocytic. However, immunocytochemical analysis indicated that the connexin43 protein was indeed associated with GFAPimmunoreactive cells (Fig. 3A,B). In contrast, similar immunolocalization of connexin43 was not detectable in C6 cells. The presence of gap junctions between astrocytes has been described morphologically, electrophysiologically and immunohistochemically [6, 9, 15, 16, 25, 281. While Dudek et al. [6] have reported the presence of a 27 kDa protein which cross-reacts with an antibody raised against liver gap junctions in cultures of primary astrocytes, our data indicate that only the heart-type connexin43 mRNA is detectable in astrocyte cultures, in agreement with previous in situ studies [5,7,28]. One of the main functions attributed to these astrocytic gap junctions concerns their role in the spatial buffering of ions by astrocytes [8, lo]. In addition, intercellular communication via gap junctions has been hypothesized to be of major importance in the regulation of cell proliferation and subsequent differentiation [14]. Loss of this coupling has been implicated in tumorigenesis [12, 141, although direct evidence for this remains to be obtained. Ultrastructural examination has revealed the presence of gap junctions in cultures of astrocytes, while these structures were not detected in cultures of C6 cells [25]. Since these cells were relatively early passage, their astrocytic phenotype would not be definitive [21]. This was re-

While punctate

is not detectable

staining

for connexin43

is apparent

in GFAP

in glioma cells. Bar = ‘50 pm.

fleeted in both the low level of GFAP expression, as previously reported by immunofluorescence [4], and the level of connexin43 mRNA and intercellular coupling. These C6 glioma cells grow rapidly when implanted into the brain, resulting in a large astrocytoma [ 1,2]. The lack of significant dye-coupling and the relatively low level of connexin43 mRNA in these cells is consistent with a low degree of intercellular coupling via gap junctions. This lack of extensive intercellular coupling may contribute to the vigorous growth of these cells in vivo. The authors are grateful to D.L. Paul, Harvard University, for connexin32 and connexin43 cDNAs, to B.J. Nicholson, SUNY at Buffalo, for connexin43 antiserum, to A.J. Tobin, UCLA, for the GFAP cDNA, to R.F. Del Maestro for C6 cells and to Mrs. Lynne Wood for technical assistance. This research was supported by grants from the Medical Research Council of Canada and the Upjohn London Neuroscience Program (UNLP 6007). C.C.G.N. is a scholar of the Medical Research Council of Canada. Auer,

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Rubinstein,

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