(Received for publication, May 19, 1995; and in revised form, July 6, 1995)
From the
Multiple cis-acting elements have been defined to be important for the transcriptional regulation of the human insulin receptor (hIR) gene expression. We report here that one of these elements also mediated the stimulation of hIR promoter activity by the retinoblastoma gene product (Rb). The cis-element responsible for Rb stimulation was localized to the GA and GC boxes situated between -643 to -607 of the hIR gene. We have previously demonstrated that these GA and GC boxes bind Sp1 with high affinity and are responsible for E1a activation of hIR promoter activity. Mutation of these sequences completely abolished Rb-dependent enhancement of hIR promoter activity. In addition, we localized three regions in the N-terminal domain of Rb to be involved in stimulation of hIR promoter activity. Our results represent one of the first studies to demonstrate a functional importance assigned to the multiple phosphorylation sites in the N terminus of Rb. Finally, the mechanism by which Rb activates the hIR promoter are presented.
Insulin plays an important role in the regulation of cellular
metabolism in addition to stimulating the growth and proliferation of
cells. It binds to the insulin receptor with high affinity and exerts
its effects by stimulating glucose, ion, and amino acid influx, and by
modifying rate-limiting enzymes involved in glucose, lipid, and protein
metabolism (Straus, 1984). Upon binding insulin, the insulin receptor
phosphorylates multiple tyrosine residues of insulin receptor
substrate-1 (IRS-1), ()which then binds to various SH2
domain-containing signal transduction proteins. The tyrosine kinase
activity of the insulin receptor is tightly associated with its
biological activity (for reviews, see Czech(1985), Kahn (1985), and
Rosen(1987)).
The human insulin receptor (hIR) gene has been isolated and characterized by different groups (Seino et al., 1989; Lee et al., 1992; Araki et al., 1987; McKeon et al., 1990; Tewari et al., 1989), and its promoter is located within 700 bp upstream of the ATG codon. Multiple transcription initiation sites were mapped within the GC-rich region of the hIR promoter (Seino et al., 1989; Tewari et al., 1989). Like other housekeeping genes, the hIR promoter lacks the TATA and CAAT boxes. It is extremely GC-rich and contains multiple GC boxes that are binding sites for the mammalian transcription factor Sp1 (Lee et al., 1992; Araki et al., 1991; McKeon et al., 1990; Kadonaga et al., 1986). There are two GA and four overlapping GC boxes at -643 to -594, and these have been shown to be important for the expression of the hIR gene (Lee et al., 1992). Mutations of the GC and GA boxes markedly reduced the transcription of the hIR gene. Gel-shift analyses showed that Sp1 binds to the cluster of four GC boxes located from -593 to -618 (Lee et al., 1992; Araki et al., 1991; Cameron et al., 1992). In addition to the GC boxes, binding sites for two novel factors, insulin receptor nuclear factors; I and II (IRNF-I and IRNF-II), have also been demonstrated to bind to the -530 to -550 and the -500 to -520 regions of the hIR promoter, respectively. Mutations that abolished IRNF-I and IRNF-II binding greatly reduced the expression of the hIR gene, indicating that both factors are important for the hIR gene expression (Lee et al., 1992). Recently, our group has demonstrated that the E1a adenoviral oncoprotein can also transactivate hIR, and this is mediated through the Sp1-binding sites (Kim et al., 1994). Loss of binding activity of Sp1 to the GA and GC boxes located between -643 to -607 results in reduction of the basal activity and the loss of E1a inducibility of the hIR gene.
Sp1 is a GC
box-specific binding protein, which activates the transcription of many
viral and cellular genes, including the SV40, epidermal growth
factor receptor, insulin-like growth factor binding protein-2, and
transforming growth factor-1 gene as well as the hIR gene (Lee et al., 1992; Dynan and Tjian, 1983; Xu et al., 1993;
Boisclair et al., 1993; Kim et al., 1991). The
structure and function of Sp1 have been extensively characterized. The
DNA-binding domain of Sp1 is located in the C terminus and consists of
three C
H
type zinc-finger motifs which bind to
the GC boxes (Hoey et al., 1993; Kadonaga et al.,
1986). There are two glutamine rich activation domains, A and B, which
have transcriptional activity (Courey et al., 1989). These
activation domains interact with the TFIID complex which consists of
the TATA-binding protein (TBP) and the multiple TBP-associated factors
(TAFIIs) (Gill and Tjian, 1992). The gene encoding TAFII-110 has been
cloned and shown to interact with the A and B domains of Sp1 to mediate
Sp1-dependent transcription (Hoey et al., 1993).
Rb has
been shown to regulate cell cycle progression and repress cell growth
and differentiation (Bookstein et al., 1990; Horowitz et
al., 1990). It has been demonstrated that Rb supresses growth
related gene expression by directly inactivate transcription factors,
including E2F, ATF-2, and Elf-1 (Bagchi et al., 1991;
Chellappan et al., 1991; Wang et al., 1993; Kim et al., 1992a, 1992b). Hypophosphorylated forms of Rb have
been shown to interact with E2F and its related family members through
the conserved Rb pocket (Buchkovich et al., 1989; Chen et
al., 1989; Pietenpol et al., 1990), and Rb-E2F
interaction inhibits the transactivation function of E2F.
Hyperphosphorylation of Rb by cyclin-cdk complexes, however, cause Rb
to release E2F which is then free to transactivate its target gene
(Bagchi et al., 1991; Chellappan et al., 1991;
Dynlacht et al., 1994). Alternatively, Rb regulates cellular
proliferation and cell cycle control by activating cellular genes,
including c-fos, TGF-1, and c-jun through a conserved cis-activating element, termed the
retinoblastoma control element (RCE). RCE are GC-rich sequences,
present in the promoter of the above genes. Sp1 binds to and stimulates
transcription through the RCE motif (Kim et al. 1991, 1992a,
1992b; Pietenpol et al., 1991; Robbins et al., 1990;
Yu et al., 1992; Chen et al., 1994). A protease
sensitive Sp1 negative regulator (Sp1-I), which inhibits Sp1 binding to
the Sp1 binding site of the c-jun promoter, has recently been
identified (Chen et al., 1994). The inhibition of Sp1 binding
by Sp1-I was reversed by the addition of bacterially expressed
recombinant Rb, suggesting that Rb may sequester Sp1-I and release Sp1
from its inhibitor. The functional domain of Rb which interacts with
the putative Sp1-I has not yet been defined.
Insulin interacts with the insulin receptor to exert its role in long term cell proliferation and growth. Since Sp1 has been shown to be important for the regulation of transcription of the hIR gene and Rb has been shown to be able to regulate cell growth through either Sp1 binding to the RCE elements or via the E2F pathway, we investigated whether Rb can regulate hIR gene expression in a human hepatoma cell line, HepG2. Our results demonstrate that Rb stimulates the expression of the hIR gene through Sp1 binding sites in the promoter. In addition overexpression of Sp1 can also augment hIR promoter activity, suggesting Sp1 can substitute for Rb to stimulate the hIR gene expression. Our results are consistent with the hypothesis that Rb can sequester Sp1-I, releasing Sp1 from the inhibitory factor Sp1-I, and allowing Sp1 to activate the hIR promoter activity. Finally and most importantly, we have defined the N-terminal domain of the Rb molecule responsible for the stimulation of the hIR gene promoter activity. This represents one of the first demonstrations of a functional role for these N-terminal regions of Rb.
Figure 1: Stimulation of hIR promoter by Rb. HepG2 cells were transfected with 5 µg of reporter constructs, phIRCAT-1819 or pSV2CAT, with or without expression vector pCMV-Rb (2 µg). Cells were harvested at 44 h. after transfection, and CAT activity assayed by TLC.
Figure 2: Localization of the Rb-response elements in the hIR promoter. The indicated 5`-deletion constructs of hIR promoter were co-transfected with or without expression vector, pCMV-Rb, into HepG2 cells. Cells were harvested 44 h. after transfection, and assayed for CAT activity. Data shown here is a summary of three independent transfection experiments. Fold of stimulation represents Rb-dependent hIR-reporter induction.
Figure 3: Effect of the GA and GC box mutation on Rb stimulation. The wild type phIR CAT-873 or the linker scanning constructs with mutations in GA and GC boxes were co-transfected with or without expression vector pCMV-Rb into HepG2 cells. Cells were harvested 44 h after transfection and assayed for CAT activity. Data shown here is a summary of three independent transfection experiments. Fold of stimulation represents Rb-dependent hIR-reporter induction.
Figure 4: Stimulation of hIR promoter by Rb mutants. Reporter construct phIRCAT-1819 were cotransfected with or without different Rb deletion mutants into HepG2 cells. These mutants are gifts of Dr. D. J. Templeton (Qian et al., 1992). The deletion end points are shown in the Fig. Cells were harvested 44 h after transfection and assayed for CAT activity. A and B represent the pocket domains of Rb which are important for T-antigen and E1a viral protein binding. Fold of stimulation represents Rb dependent increase of hIR-reporter activity. Three separate experiments were carried out with duplicates, and the results are expressed as means ± S.D. Statistical significance were determined by performing analysis of variance using Fisher's post-hoc test. Fold of stimulation obtained with d1, d2, d7, and d10 are significantly lower than the fold of stimulation obtained with the wild type Rb (p = 0.005).
Figure 5: Stimulation of hIR promoter by Sp1 and Rb. 2 µg of either Rb or Sp1 expression vector, pCMV-Rb or pCMV-Sp1, were co-transfected with 5 µg of the wild type phIR CAT-873 or the linker scanning constructs with mutations in GA and GC boxes into HepG2 cells. PCMV control plasmid have been used in the control co-transfection with hIR constructs.
Figure 6:
a, dose-dependent activation of hIR by
Sp1. Reporter construct phIRCAT-1819 was co-transfected with increasing
amount of expression vector pCMV-Sp1 into HepG2 cells in the presence
() or absence (
) of pCMV-Rb. b, dose-dependent
activation of hIR by Rb. Reporter construct phIRCAT-1819 was
co-transfected with increasing amount of expression vector pCMV-Rb into
HepG2 cells in the presence (
) or absence (
) of
pCMV-Sp1.
Rb is a 110-kDa nuclear protein that is modified by cell cycle-regulated hyperphosphorylations (Bandara et al., 1991; Buchkovich et al., 1989; Chen et al., 1989). Mutations in the RB-1 gene have been observed in a wide variety of tumors including retinoblastoma, osteosarcomas, bladder carcinomas, small-cell lung carcinomas, prostate carcinomas, and cervical carcinomas. The wide variety of tumors carrying mutated RB-1 genes suggest that Rb has an important role in the regulation of normal cell proliferation (for review, see Lees et al.(1991), Weinberg (1992), and Horowitz et al.(1990)). It has also been reported that functional replacement of the RB-1 gene can suppress tumorigenic phenotypes and inhibit growth in proliferating cells (Huang et al., 1991; Bookstein et al., 1990; Sumegi et al., 1990; Takahashi et al., 1991; Templeton et al., 1991). Rb has been shown to be able to complex with the oncoproteins encoded by several DNA tumor viruses, including adenovirus E1a, simian virus 40 T antigen, and human papillomavirus 7 (DeCaprio et al., 1989; Dyson et al., 1989; Whyte et al., 1988). Recently, Rb has also been shown to bind to the cellular E2F transcription factor, and repress E2F-mediated gene expression thereby suppressing cellular proliferation. The cell cycle regulated hyper-phosphorylated form of Rb, however, can no longer associate with E2F, and thereby loses the ability to repress E2F-regulated transactivation (Bagchi et al., 1991; Dynlacht et al., 1994; Buchkovich et al., 1989; Chen et al., 1989; Pietenpol et al., 1990). These results suggested that Rb has a very important role in regulating cell cycle progression and in repressing cell growth and differentiation.
Another target of Rb is the Sp1 family of transcription factors
which binds to the RCE and activate RCE containing genes. Robbins et al.(1990) demonstrated that in the c-fos promoter,
Rb can repress the transcription of c-fos through the RCE and
reduce the AP-1 stimulatory activity. Similarly, Kim et al. (1992a) showed that RCE-like sequences are important for positive
regulation of insulin-like growth factor II (IGF-II) gene by human Rb.
Furthermore, they have shown that the TGF-1 and c-fos promoters can both be positively and negatively regulated by Rb
through the same RCE element in different cell types (Kim et
al., 1992b). Subsequently, they demonstrated that Rb regulates the
expression of IGF-II through Sp1 (Kim et al., 1992b),
and that Sp1 binding to DNA is not required, since a GAL4-Sp1 fusion
protein can also confer the transcriptional regulation by Rb.
We
have shown here that Rb can stimulate the expression of the hIR gene by
5-6-fold. Rb-dependent stimulation is mediated through sequences
between -643 and -607, which contains two GA and three GC
boxes through which Sp1 binds. By either deleting the GC-rich sequences
between -643 and -607 or mutating the GC boxes for Sp1
binding in this region, we have demonstrated that the Rb-dependent
stimulation is eliminated. Our results indicate that the Rb-dependent
stimulation of the hIR promoter is mediated through Sp1-binding sites.
These results are similar to those observed with the c-jun and
TGF-1 promoters (Kim et al., 1991; 1992a; Chen et
al., 1994).
Recently, Chen et al.(1994) showed that Rb
can stimulate the c-jun promoter activity. A 20-kDa protein
has been implicated to have the ability to prevent the binding of Sp1
to DNA. This Sp1 inhibitor (Sp1-I) is a heat-labile and proteinase
K-sensitive protein, which presumably interacts with both Sp1 and Rb.
When it binds to Sp1, it prevents Sp1 from binding to DNA, hence
inhibiting the ability of Sp1 to transactivate target genes. In its
presence, Rb competes with Sp1 for the Sp1-I binding, thus releasing
Sp1 and allowing it to bind to the GC boxes and transactivate target
genes. For the hIR, we have not been able to detect the direct
association of Rb with Sp1 by immunoprecipitation and supershifts in
gel mobility assays, suggesting that Rb stimulation of the hIR promoter
is not through direct interaction with Sp1. Therefore, it
is likely that Sp1-I, similar to the c-jun system (Chen et
al., 1994), interacts with Rb and releases Sp1 for the hIR gene
stimulation. If this is the case, overexpression of Sp1 should be able
to replace Rb in the hIR promoter stimulation. Indeed as shown in Fig. 5, overexpression of Sp1 could substitute for Rb as it
enhances the hIR expression to a similar extent as the Rb-dependent
activation. This is consistent with the notion that Rb sequesters Sp1-I
and releases Sp1 from its inhibition, leading to the activation of the
hIR promoter activity. This conclusion is further supported by the
failure of Rb to further stimulate the hIR promoter activity at a
saturating amount of Sp1 (Fig. 6). Thus, the enhancement of hIR
promoter activity by Rb and Sp1 is likely mediated through the same
pathway; and the role of Rb is to sequester Sp1-I, removing it from
inhibition of the activity of Sp1.
Rb contains multiple functional domains, including two discontiguous regions, the A and B pockets, and a N- and a C-terminal domains. The A and B pockets, located at amino acids 389-580 and 614-775, are required for interactions with DNA tumor virus antigens, E1a, and transcription factor E2F (Qian et al. 1992; Hu et al., 1990; Kaelin, et al., 1990). The N-terminal domain consists of three regions which contain kinase recognition sites (Qian et al., 1992; Qin et al., 1992) and has been proposed to be important for hyperphosphorylation of Rb (Qian et al., 1992). Although this domain has been implicated to be involved in growth suppression, a biological function for this region has not been well defined (Karantza et al., 1993; Hogg et al. 1993; Dryja et al., 1993). We have shown here that the N-terminal regions (amino acids 37-89, 89-140, and 343-389) are important for stimulation of hIR promoter activity. In addition, the sequences between the A and B pockets (amino acids 580-614) which contains putative phosphorylation sites are also important for the stimulation. In contrast, the A and B pockets, which are important for interaction with E2F are not essential for activation of hIR promoter activity. These results are consistent with the hypothesis that the Rb stimulation of hIR is mediated through sequestering Sp1-I rather than binding to E2F. Most importantly, we have defined the N-terminal region of Rb to be involved in activation of hIR promoter activity. This represents one of the first assigned biological function for this region.