(Received for publication, December 22, 1994; and in revised form, January 24, 1995)
From the
To examine the potential impact of disrupting
``pocket'' protein function on cardiac differentiation and
growth, we introduced 12 S E1A genes into neonatal ventricular
myocytes, by adenoviral gene transfer. In the absence of E1B, E1A was
cytotoxic, with features typical of apoptosis. In the presence of E1B,
E1A preferentially inhibited transcription of cardiac-restricted
-actin promoters, and reactivated DNA synthesis in cardiac
myocytes, without cell death. Mutations that abrogate known activities
of the amino terminus of E1A, versus conserved region 2,
demonstrate that the ``pocket'' protein- and p300-binding
domains each suffice, in the absence of the other, for transcriptional
repression and re-entry into S phase.
Though the molecular events governing cell-cycle exit in cardiac
muscle are cryptic at present, potential clues are available from
skeletal muscle and other model systems. Mitogenic growth factors
control the cell cycle in part through cyclin-dependent protein
kinases, which in turn alter the phosphorylation or expression of
so-called ``pocket'' proteins including the retinoblastoma
gene product, Rb, ()p107, and
p130(1, 2, 3) . Hyperphosphorylation or
down-regulation of pocket proteins causes release of the transcription
factor E2F, activating E2F-dependent genes needed for DNA
synthesis(1) . A concurrent role for pocket proteins, as a
platform for the action of myogenic transcription factors, suggests
that release of myogenic factors from the pocket abrogates their
function, repressing muscle-specific
transcription(2, 3) .
Certain transforming proteins of DNA tumor viruses including adenovirus E1A, SV40 large T antigen, and human papilloma virus E7 physically interact with pocket proteins and can be used as surrogates for growth factors, to establish the involvement of pocket proteins in cells' growth or differentiation(1, 2, 3, 4, 5, 6, 7) . Evidence for operation of a pocket protein-dependent pathway in cardiac muscle is provided by transgenic mice that express SV40 large T antigen in myocardium, which provide a conceptual framework for our investigations(8, 9, 10) . However, large T antigen also interacts with targets distinct from pocket proteins, including the tumor suppressor p53, and systematic mutagenesis to discriminate among these is impracticable in vivo. Moreover, since the cardiac promoters used are first transcribed early in cardiogenesis, transgenic models might not predict the outcome of de novo interventions, after birth.
As a step toward identifying positive and negative regulators of myocardial growth, we have investigated the consequences of E1A proteins in neonatal ventricular myocytes, cells that still retain limited ability to respond to exogenous growth signals(11) . We chose to use E1A, given the precedent of E1A's impact on skeletal muscle (4, 5, 6) and the delineation of E1A domains for interaction with cellular targets(7, 12, 13) . Two major E1A transcripts, 13 S and 12 S, yield proteins of 289 and 243 amino acids. Conserved region (CR) 1, CR2, and CR3, shared across adenovirus serotypes, and the amino terminus are essential for protein binding and biological activity. CR3 encodes a domain for transactivation of late viral genes necessary for virus replication, absent from 12 S E1A products. One active site, for binding pocket proteins, comprises CR2 together with CR1(7, 13) ; a second, comprising the amino terminus of E1A with CR1, binds p300, a bromodomain protein with properties of a transcriptional coactivator (14) . A missense mutation of CR2 and the amino terminus, R2G,C124G, abrogates binding both to pocket proteins and p300(7, 13) , and disrupts all known E1A functions. Selective mutations that disrupt binding to pocket proteins but not p300, or vice versa, can test each pathway individually(7, 12, 13) . Finally, adenoviruses ensure virtually uniform gene transfer to cardiac myocytes, an advantage for efforts to manipulate myocyte growth itself(15) .
Preliminary plasmid transfections revealed dose-dependent
repression of skeletal and cardiac -actin promoters by wild type
12 S E1A, and not R2G,C124G(
); however, inhibition of
constitutive promoters was equivalent in extent, suggesting a
generalized phenomenon. Although little attention was given to
programmed cell death in connection with E1A in striated muscle,
recently it has become appreciated that E1A unaccompanied by E1B can
lead to apoptosis(26) . To test whether these pilot results
were confounded by the absence of E1B, we transfected cardiac myocytes
with the skeletal actin reporter, 12 S E1A, and increasing
concentrations of E1B expression vector.
E1B itself had no
effect on the skeletal actin promoter but partially restored
E1A-inhibited transcription (p < 0.01). Thus, repression by
E1A was due in part to the absence of E1B, and hence, potentially,
apoptosis.
To circumvent this impediment, we employed recombinant
adenoviruses that coexpress 12 S E1A in tandem with E1B. Except where
noted, cultures were incubated with 20 plaque-forming units/cell, from
24 to 30 h after plating, and were maintained in serum-free
medium(18) . This multiplicity of infection achieves gene
delivery to 95% of neonatal ventricular cells.
To verify that E1A provokes cardiac myocyte death in the absence of E1B, ventricular myocytes were stained 24-72 h after infection with calcein acetoxymethyl ester and ethidium homodimer-1 to mark live and dead cells, respectively (Fig. 1). At 24 h, cells infected with 12 S E1A + E1B or R2G,C124G + E1B were indistinguishable from uninfected cells; 12 S E1A - E1B increased cell death 4-5-fold (p < 0.01). Likewise, cell death at 72 h did not differ significantly between uninfected cells and those infected with 12 S E1A + E1B (p = 0.44), whereas cell death was increased 5-fold by 12 S E1A - E1B (p = 0.002, versus uninfected cells). To confirm that E1A in the absence of E1B was cytotoxic for cardiac myocytes themselves, we utilized indirect immunofluorescence for sarcomeric MHC (22) and Hoechst dye 33258 for nuclear DNA. No difference from uninfected cells was seen with 12 S E1A + E1B (Fig. 2, A-F; Table 1). By contrast, cardiac myocytes infected with 12 S E1A - E1B showed cell rounding and disruption of nuclear DNA (Fig. 2, G-I). To corroborate that apoptosis was the mechanism of cell death induced by E1A, we tested for nucleosomal DNA fragmentation (Fig. 2J). Laddering was induced by 12 S E1A - E1B, but not 12 S E1A + E1B, consistent with the proportions of live and dead cells.
Figure 1:
E1A, in the absence of E1B, induces
cell death in cardiac myocytes. After infection, cells cultured for 24 (A-D) or 72 (E and F) h (18) were visualized using calcein acetoxymethyl ester (green) versus ethidium homodimer-1 (red). A, uninfected cells; B, R2G,C124G + E1B; C and E, 12 S E1A + E1B; D and F, 12
S E1A - E1B. Bar, 80 µm. Lower panel,
results at 24 h were calculated using 200 cells for each condition
shown.
Figure 2: E1A-induced nuclear fragmentation in cardiac myocytes, in the absence of E1B. A-C, uninfected cells; D-F, 12 S E1A + E1B; G-I, 12 S E1A - E1B. A, D, and G, phase-contrast microscopy; B, E, and H, sarcomeric MHC immunofluorescence; C, F, and I, Hoechst dye 33258. Cells infected with 12 S E1A + E1B indicate the dependence of cytotoxicity at 48 h on the absence of E1B. Bar, 10 µm. J, DNA laddering induced by 12 S E1A - E1B.
To reexamine
transcriptional repression by E1A in this context, cardiac myocytes
were subjected to conventional transfection with reporter plasmids,
immediately after incubation with virus. Luciferase and lacZ
activities were determined 48 h later (Fig. 3A; Table 1). The endogenous cardiac and skeletal -actin genes
are co-expressed at this stage in the rat both in vivo(27) and under these conditions of cell culture (18) . Transcription of the skeletal
-actin promoter was
reduced to 15 ± 2% of control by wild-type 12 S E1A + E1B
and was not affected by R2G,C124G + E1B, despite similar levels of
E1A protein by Western blot analysis.
Mutations that
interfere with p300 binding alone (R2G + E1B), or pocket protein
binding alone (Y47H,C124G + E1B), retained the ability to repress
skeletal actin transcription (27 ± 5% and 16 ± 2%,
respectively; p < 0.01 versus control cells).
Comparable sensitivity to 12 S E1A + E1B was seen for the cardiac
-actin promoter (19 ± 2% of control cells), whereas the
cytoplasmic
-actin and herpes simplex virus thymidine kinase
promoters were much less sensitive (
50% relative to the control; p < 0.01 for each, versus the sarcomeric
-actin promoters).
Figure 3:
E1A preferentially represses
cardiac-restricted gene transcription (A) and reactivates DNA
synthesis in cardiac muscle cells (B). A, luciferase
activity is corrected for lacZ (mean ± S.E.) and is
expressed relative to that of each promoter, respectively, in
uninfected control cells. SkA, skeletal -actin; CaA, cardiac
-actin; tk, herpes simplex virus
thymidine kinase;
Ac, cytoplasmic
-actin. B, DNA synthesis was assessed by immunoperoxidase staining for
BrdUrd. See Fig. 4for details and representative images. MOI, multiplicity of infection.
Figure 4: E1A-dependent DNA synthesis in cardiac muscle cells. Cells were cultured in serum-free medium, supplemented with 10 µM BrdUrd 44-48 h after exposure to virus. A, uninfected cells; B and G, 12 S E1A + E1B; C, 12 S E1A - E1B; D and H, R2G,C124G + E1B; E, R2G + E1B; F, Y47H,C124G + E1B. A-F, immunoperoxidase detection of BrdUrd. G and H, immunofluorescence confocal microscopy for BrdUrd (fluorescein) and sarcomeric MHC (rhodamine). BrdUrd-positive, MHC-positive cells are seen in panelH. Note the persistence of sarcomeric MHC as a marker of cell identity at this time-point. I, superposition of Hoechst 33258 and MF20 immunofluorescence in cardiac fibroblasts. No sarcomeric MHC was detected. Bar, 50 (A-F) or 20 (G-I) µm.
To determine whether E1A can reactivate DNA
synthesis in cardiac muscle cells, and if so, which domains suffice,
DNA synthesis was measured by immunoperoxidase staining for BrdUrd (Fig. 3B and 4, A-F; Table 1);
preliminary studies with [H]thymidine established
that maximal DNA synthesis in the ventricular cells occurred
44-48 h after infection with 12 S E1A + E1B.
12
S E1A + E1B provoked DNA synthesis between 44 and 48 h in up to
80% of neonatal cells; multiplicities of infection from 20 to 200 did
not differ significantly. R2G,C124G + E1B was inactive, whereas
mutations that bind either pocket proteins or p300 were both sufficient
for DNA synthesis. At 48 h, no increase in cell number was evident, as
expected from the fact that the majority of cells were in S phase. No
increase in cell number was apparent even at 72 h, regardless of the
virus used (except 12 S E1A - E1B, which reduced cell number more
than 50%). Hence, E1A was not sufficient to trigger proliferation under
these serum-free conditions (cf. (28) ).
To
substantiate the conclusion that E1A reactivates DNA synthesis in
cardiac myocytes, we performed double immunocytochemistry for
sarcomeric MHC and BrdUrd (Fig. 4, G and H).
Since myosin staining is excluded from the nucleus (e.g.Fig. 4H), comparing rhodamine and fluorescein
fluorescence established for each myosin-positive cell that the
BrdUrd-positive nucleus was that of the myocyte itself, not a
superimposed fibroblast. Induction of DNA synthesis in cardiac myocytes
was confirmed for R2G + E1B and Y47H,C124G + E1B. Thus, cardiac myocytes are susceptible to reinduction of DNA
synthesis by each of these complementary E1A proteins. The half-life of
cardiac MHCs ranges from 2 to 5 days, depending on the protocol and
preparation(29, 30) ; consequently, persistence of MHC
protein is not inconsistent with repression of transcription at the
time points we studied.
For both transcription repression and reactivation of DNA synthesis, the activity of E1A mutations in cardiac muscle was consistent with operation of two alternative pathways, potentially dependent on pocket proteins and p300, respectively. This interpretation is provisional, as protein-protein interactions are not demonstrated here and differences from other cell types might exist. Preferential inhibition of lineage-specific genes, superimposed on significant but lesser inhibition of nominally constitutive genes, was characteristic of E1A in skeletal muscle as well(6, 31, 32) , concordant with reports of generalized repression by E1A (33) (cf.(4) and (5) ). Although E1A can reportedly sequester the TATA box-binding complex, holo-TFIID(34) , this mechanism is unlikely to account for our results, as the interaction is chiefly mediated by CR3, which is deleted in all 12 S E1A proteins; alternatively, 12 S E1A may also bind TATA-binding protein, weakly by comparison to 13 S E1A(35) . Considering the proposal that transcriptional repression by E1A is mediated by p53 (36) , inhibition of ``constitutive'' promoters may reflect less than complete relief of p53 by our co-expressed E1B gene.
In neonatal cardiac cells, p107 is the predominant pocket protein transcript and is subsequently down-regulated in parallel with p53 (37) ; Rb protein also is expressed in the heart(37) . The corresponding proteins have been presumed to be present at this stage of cardiac maturation. Although the spatial and temporal expression of p300 are unknown, the capacity to bind either pocket proteins or p300 was sufficient in the present study to repress transcription and elicit DNA synthesis in ventricular muscle cells. Thus, the missense mutations employed here perhaps explain the reported need for CR1, yet not CR2, in repression of skeletal muscle-specific genes(4) : deletion of CR1 abrogates binding both to p300 and all known pocket proteins, whereas deletion of CR2 still permits binding to p300(38) . Hence, a p300-dependent pathway may regulate lineage-specific transcription, at least in striated muscle, more generally (cf.(39) ). To our knowledge, the experiments reported here provide the first indication of p300 function in the cardiac muscle lineage. Given that E1A can reactivate DNA synthesis even in terminally differentiated neurons, it will be of interest to the exploit the ability of recombinant adenoviruses to deliver E1A genes to ``post-mitotic'' adult ventricular myocytes(15) .