(Received for publication, April 20, 1995; and in revised form, July 21, 1995)
From the
We transfected the cDNA for the cell-to-cell channel protein connexin-43 (Cx43) into Morris hepatoma H5123 cells, which express little Cx43 and lack gap junctional communication (open cell-to-cell channels). We found that cells overexpressing Cx43 nonetheless lacked open cell-to-cell channels, but that inhibition of glycosylation by tunicamycin induced open channels in these cells. Tunicamycin also induced biochemical changes in Cx43 protein; the level increased, and a considerable fraction became phosphorylated and Triton X-100 insoluble, in contrast to untreated cells where Cx43 was non-phosphorylated and Triton X-100 soluble. Although tunicamycin caused the formation of open channels, channels were not found aggregated into gap junctional plaques, as they are when they have been induced by elevation of intracellular cAMP. The results suggest that although Cx43 itself is not glycosylated, other glycosylated proteins influence Cx43 posttranslational modification and the formation of Cx43 cell-to-cell channels.
Cell-to-cell channels mediate intercellular communication by
providing a direct pathway for the exchange of molecules up to
1-2 kDa (Schwarzmann et al., 1981). The molecules
transferred include signaling molecules, which may play important roles
in tissue homeostasis (Loewenstein, 1981), cell growth control
(Loewenstein and Rose, 1992), and embryonic development (Warner, 1992).
The channels are known to cluster into often quite large aggregates,
forming the so-called gap junctions. In electron microscopic images of
freeze-fractured gap junctions, the channels show up as particles of
uniform size that span the two adjoining membranes (Kreutziger, 1968;
Goodenough and Revel, 1970). The channels are formed from membrane
proteins called connexins (Beyer et al., 1990; Kumar and
Gilula, 1992). More than a dozen different connexins have been
identified in vertebrates, all of which share similar topology and
amino acid sequence (Kumar and Gilula, 1992). One well studied and
widely expressed connexin is connexin-43 (Cx43), ()found in
the heart (Beyer et al., 1987) and many other tissues (Beyer et al., 1987; Micevych and Abelson, 1991) as well as in a
number of established cell lines (Musil et al., 1990; Mehta et al., 1992).
Progress has been made in understanding how cell-to-cell channels are formed. For example, it is now known that after synthesis, Cx43 (and probably the other connexins, too, see Rahman et al.(1993)) is first assembled into multimeres called connexons or hemichannels in the Golgi (Musil and Goodenough, 1993); the hemichannels are then transported to the plasma membrane where they find counterparts on the adjoining cell's membrane to form the cell-to-cell channels proper. Yet, little is known about how the cell-to-cell channels are concentrated into the gap junction plaques. In some cells, a phosphorylated and Triton X-100 insoluble form of Cx43, but not its non-phosphorylated and Triton X-100 soluble form, is localized in the gap junctional plaques (Musil and Goodenough, 1991). It is not clear whether Cx43 becomes Triton X-100 insoluble as a consequence of hemichannel interlocking, i.e. channel formation, or as a consequence of the clustering into gap junction plaques. Neither is it clear what role phosphorylation has in Cx43 channel or gap junction formation.
We have found previously that inhibition of glycosylation by tunicamycin (Tm) greatly increases the formation of open channels in a variety of Cx43-expressing cells (Wang and Mehta, 1995). In the present study, we investigate whether any biochemical changes in Cx43 are associated with Tm-induced channel formation. For this, we constitutively expressed Cx43 in a cell line that lacks open cell-to-cell channels and studied the effects of tunicamycin on cell communication via Cx43 channels and on phosphorylation, Triton X-100 solubility, and cellular localization of Cx43.
The expression of Cx43 protein in MHD1 cells and in one Cx43 overexpressing clone, MHD1-43A, is shown in Fig. 1A. The MHD1-43A cells express Cx43 abundantly, manyfold higher than the parental MHD1 cells. And like in the parental cells, Cx43 in the MHD1-43A cells is mainly in the non-phosphorylated form, appearing as one major band of 42 kDa on SDS-PAGE (Fig. 1A, lane 2; see also Fig. 1C, lane 1).
Figure 1:
Western blot analysis of Cx43 in MHD1
and MHD1-43A cells. A, overexpression of Cx43 in Morris
hepatoma cells. Lane 1, MHD1; lane 2, MHD1-43A. B, effect of Tm on Cx43 in MHD1 cells. Lane 1,
control (0.4% MeSO, 8 h); lane 2, Tm-treated cells
(4 µg/ml, 8 h). C, effect of Tm on Cx43 in MHD1-43A
cells. Lane 1, control; lane 2, Tm. D,
dephosphorylation of Cx43 from Tm-treated MHD1-43A cells by
alkaline phosphatase. Lysates treated with (lane 1) and
without (lane 2) alkaline phosphatase. The positions of
molecular mass markers (in kDa) are indicated on the left for
Western blots A and B and on the right for
Western blots C and D (30 µg total
protein/lane).
Figure 2:
Tunicamycin induces gap junctional
communication in Cx43 overexpressor cells. A, cell-cell
transfer of Lucifer Yellow in MHD1-43A cells. panels a and b, control (0.4% MeSO, 8 h). Panels c and d, tunicamycin (4 µg/ml, 8 h). Phase contrast (a, c) and fluorescent (b, d)
images are shown. Photographs were taken 5 min after injection of
Lucifer Yellow into the asterisk-marked cells. B,
time course of the effect of tunicamycin and cycloheximide on
communication. Each data point represents the mean ±
S.E. from 30-50 injections in three experiments. Tm, 4 µg/ml;
cycloheximide (CHX), 60 µg/ml.
Figure 3: Reduction of lectin binding after tunicamycin treatment. Cell surface binding of rhodamine-labeled wheat germ agglutinin (a and b) and D. biflorus agglutinin (c and d) in control (a and c) and tunicamycin-treated (b and d) MHD1-43A cells.
Tunicamycin has been reported to inhibit protein synthesis (Elbein, 1987), and inhibition of general protein synthesis may somehow increase communication (Azarnia et al., 1981). To test whether tunicamycin's effect on communication could be due to general inhibition of protein synthesis, we treated MHD1-43A cells with the protein synthesis inhibitor, cycloheximide. As seen in Fig. 2B, communication changed little, showing that the effect of tunicamycin cannot be explained by general inhibition of protein synthesis.
Under control conditions, Cx43 in the non-communicating MHD1 cells was predominantly Triton X-100 soluble (Fig. 4, lanes 1-3); Triton X-100 insoluble Cx43 was barely detectable, and this was so also after Tm treatment (Fig. 4, lanes 4-6). In contrast, forskolin treatment, which increases Cx43 expression and induces communication as well as Cx43 phosphorylation in MHD1 cells (Wang and Mehta, 1995), induced Triton X-100 insoluble Cx43 (Fig. 4, lanes 7-9). This Triton-insoluble fraction included both phosphorylated and non-phosphorylated Cx43, while the soluble fraction consisted of non-phosphorylated Cx43.
Figure 4: Western blot analysis of Triton X-100 solubility of Cx43 in MHD1 cells. Total (T), soluble (S), and insoluble (I) Cx43 of control (lanes 1-3), tunicamycin (lanes 4-6; 4 µg/ml, 8 h), and forskolin (lanes 7-9; 20 µM forskolin plus 50 µM phosphodiesterase inhibitor, Ro-20-1724, 8 h) treated cells. The positions of molecular mass markers are indicated on the left in kDa.
The Cx43 in untreated, basically non-communicating MHD1-43A and MHD1-43B cells was also mainly Triton X-100 soluble (Fig. 5, lanes 1-3 and 7-9). But after tunicamycin treatment, with the appearance of open channels and of phosphorylated Cx43 (the two upper bands in lanes 4, 6, 10, and 12 of Fig. 5), part of the Cx43 became Triton X-100 insoluble. As in the forskolin-treated MHD1 cells, this insoluble Cx43 consisted of both non-phosphorylated and phosphorylated forms of Cx43 (Fig. 5, lanes 6 and 12), while the soluble Cx43 was mainly non-phosphorylated (Fig. 5, lanes 5 and 11).
Figure 5: Tunicamycin induces Triton-insoluble Cx43 in Cx43 overexpressor cells. Western blot analysis (same procedure as in Fig. 4) of total, Triton-soluble, and Triton-insoluble Cx43 in MHD1-43A and MHD1-43B cells with or without tunicamycin treatment is shown. Lanes 1-3, MHD1-43A control cells; lanes 4-6, tunicamycin-treated MHD1-43A cells; lanes 7-9: MHD1-43B control cells; lanes 10-12, tunicamycin-treated MHD1-43B cells. The positions of molecular mass markers are indicated on the left in kDa. Note: do not compare band intensities of Fig. 4and Fig. 5; relative intensities of bands should only be compared within one and the same blot.
Figure 6: Immunostaining of Cx43 in MHD1 (a-c), MHD1-43A (d-f), and MHD1-43B cells (g-i). Control (a, d, g), tunicamycin (b, e, h; 4 µg/ml, 8 h), and forskolin (c, f, i; 20 µM forskolin plus 50 µM Ro-20-1724, 8 h) treated cells are shown.
The lack of punctate staining is not due to an intrinsic incapability of the cells to cluster cell-to-cell channels into gap junction plaques; bright Cx43 plaque staining appeared in both MHD1 and overexpressor cells after an 8-h forskolin treatment (Fig. 6, c, f, and i). This staining is much stronger and more abundant in overexpressor cells than in parental MHD1 cells, consistent with the much higher level of Cx43 protein (Fig. 1A) and of forskolin-induced communication (data not shown) in the overexpressor cells.
In the present study, we transfected cx43 cDNA into cells that lack open cell-to-cell channels and express little Cx43 and found that cells which overexpressed Cx43 nonetheless had few open channels. The lack of open channels in the Cx43 overexpressors seems not due to any null mutation in the cx43 cDNA. The ability of cDNA-derived Cx43 to form open cell-to-cell channels is clearly evident from the difference in communication between the parental MHD1 and the overexpressor cells after tunicamycin treatment; extensive cell-to-cell transfer of tracer was induced in Cx43 overexpressing cells (Fig. 2) but not in parental MHD1 cells (Wang and Mehta, 1995). Instead, the failure of the exogenous Cx43 to make open channels points to some cellular condition non-permissive for Cx43 to make open channels, and inhibition of glycosylation remedies this condition, allowing channel formation.
Due to the lack of potential glycosylation sites in their extracellular loops, connexins are unlikely to be glycosylated. Connexin-32 is known not to be glycosylated (Hertzberg and Gilula, 1979; Rahman et al., 1993), and our result of a lack of decrease in apparent molecular mass after tunicamycin treatment confirmed that Cx43 is not glycosylated either. Therefore, a reduction of carbohydrates from cell surface proteins other than Cx43 is a more likely cause for the observed increase in communication.
From a priori considerations, glycoproteins on the cell surface can be expected to impose an inhibitory effect on the formation of cell-to-cell channels and gap junctions (Peracchia, 1985; Abney et al., 1987). It was shown previously that lectins induced or fostered intercellular communication in Aplysia neurons (Lin and Levitan, 1987) or in Xenopus oocytes (Levine et al., 1991), presumably by removing bulky glycoprotein from the plasma membrane, and we have shown that inhibition of glycosylation increased intercellular communication in a variety of mammalian cells (Wang and Mehta, 1995).
Carbohydrates may interfere with any one of the steps occurring on the membrane during the formation of open cell-to-cell channels and thereby result in decreased communication. The extracellular domain of connexins is no larger than 8-10 Å, smaller than that of many membrane glycoproteins. One possibility therefore is that large membrane glycoproteins interfere with hemichannel interlocking by hindering two adjoining plasma membranes to come close enough to allow hemichannel interaction. Little is known about how the hemichannels get to the cell-cell contact sites and how channels become concentrated in the junctional plaques. It is possible that hemichannels are transported onto the plasma membrane at random sites and then laterally diffuse to the cell-cell contacts; or, they could be directly inserted into these sites. Bulky membrane glycoproteins may impede the lateral movement of hemichannels on the plasma membrane, or the insertion of hemichannels into the plasma membrane at random or specific sites may involve some glycoprotein(s). Yet another possibility is that even when channels are formed, bulky surface carbohydrates in the immediate vicinity of channels produce some condition unfavorable for the channels to be in the open state.
Although tunicamycin treatment elevated the total Cx43 level in Cx43 overexpressors, it is unlikely that this caused the dramatic rise in communication. Instead, the greater total Cx43 protein may reflect a higher stability of Cx43 protein in channels than in hemichannels. We therefore interpret the increase in Cx43 to be the consequence of hemichannel interlocking rather than the cause of it. In agreement with this interpretation is that in the parental MHD1 cells, where tunicamycin did not induce channel formation, the Cx43 protein level did not rise; in fact, it was slightly diminished. This is consistent with the reported inhibition of protein synthesis by tunicamycin in other cells (Elbein, 1987).
One unexpected result of this study is that tunicamycin treatment induced Cx43 phosphorylation in the Cx43 overexpressor cells, raising the possibility that tunicamycin activates a kinase. Activation of protein kinase A up-regulates junctional communication and induces Cx43 phosphorylation in a variety of cells, including MHD1 cells (Wang and Mehta, 1995). But because protein kinase A activation has other effects not seen with tunicamycin treatment, e.g. stimulation of Cx43 transcription (Wang and Mehta, 1995), it is unlikely that tunicamycin activates protein kinase A. It is even less likely that tunicamycin activates protein kinase C or a tyrosine kinase because, although these kinases cause Cx43 phosphorylation, they inhibit communication (Crow et al., 1990; Filson et al., 1990; Brisette et al., 1991; Berthoud et al., 1992; Kanemitsu and Lau, 1993), whereas tunicamycin increases communication. It is therefore unclear how Cx43 gets phosphorylated after tunicamycin treatment. Cx43 hemichannels are probably not phosphorylated (Musil and Goodenough, 1993) and are in a closed conformation. When they interlock, they must undergo a conformation change, which enables them to switch to an open state. It is possible that after the conformation change, Cx43 becomes a substrate of an unidentified, constitutively active kinase and thus gets phosphorylated. The function of this Cx43 phosphorylation is not clear. There is no evidence that phosphorylation is a prerequisite for channels to open.
Musil and Goodenough(1991) showed in several cell lines that a certain form of phosphorylated and Triton X-100 insoluble Cx43 is correlated with its localization at the junctional plaques. We found this to be true also in MHD1 cells, where forskolin induced the appearance of junctional plaques concurrently with Cx43 phosphorylation and Triton X-100 insolubility. However, we noticed differences. In the cells used by Musil and Goodenough(1991), the Triton X-100 insoluble fraction contained primarily phosphorylated Cx43, while in MHD1 and Cx43 overexpressors, the Triton-insoluble fraction contained additionally a substantial amount of non-phosphorylated Cx43. Another difference is that in the overexpressor cells, after tunicamycin had induced functional channels, phosphorylated and Triton X-100 insoluble Cx43 was found, but junctional plaques were not seen. The latter result would imply that Cx43 phosphorylation and Triton X-100 insolubility correlate better with hemichannel interlocking or cell-to-cell channel formation than with channel clustering into gap junction plaques, at least not with plaques large enough to be resolved by immunostaining. The clustering of channels into plaques that are detectable by immunostaining seems to require elevation of cAMP, both in the MHD1 and the Cx43 overexpressor cells.