Glucocorticoid Inhibition of Fibroblast Proliferation and Regulation of the Cyclin Kinase Inhibitor p21Cip1

Arivudainambi Ramalingam, Aki Hirai and E. Aubrey Thompson

Department of Human Biological Chemistry and Genetics University of Texas Medical Branch Galveston, Texas 77550-0645


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids inhibit the proliferation of fibroblastic cells in vivo and in culture; however, the molecular mechanism that accounts for this effect has remained obscure. We have undertaken to elucidate the mechanism whereby glucocorticoids decrease the rate of proliferation of mouse L929 fibroblastic cells. Addition of dexamethasone to mid-log phase fibroblasts prolongs G1 phase. This increase in the G1 interval is associated with, and probably due to, inhibition of phosphorylation of the product of the Rb-1 tumor suppressor gene, pRb. Inhibition of pRb phosphorylation by cyclin D-dependent kinases can be demonstrated in vitro. Nevertheless, there is no detectable change in the expression of cyclin D1, cyclin D2, or cyclin D3. Cyclin-dependent kinase-4 (Cdk4) and Cdk6 are not down-regulated in L929 cells after addition of glucocorticoids, and the abundance of cyclin D/Cdk4 complexes does not change. Inhibition of pRb kinase activity is associated with an increase in the abundance of one of the Cdk inhibitors, p21Cip1. The abundance of another cyclin kinase inhibitor, p27Kip1, remains constant. The amount of Cdk4 that is bound to p21Cip1 increases rapidly after addition of dexamethasone, and the activity of Cdk4-pRb kinase decreases in parallel. These results indicate that glucocorticoid inhibition of fibroblast proliferation is due to induction of p21Cip1, which binds to and inactivates cyclinD/Cdk4 complexes. The abundance of p21 mRNA increases about 5-fold within 2 h after addition of dexamethasone. This effect does not obtain in L929 mutants that are null for the glucocorticoid receptor, and a variant that expresses the glucocorticoid receptor from a tetracycline-repressible expression vector demonstrates induction of p21 mRNA only in the absence of tetracycline. Cycloheximide does not block induction of p21 mRNA, and dexamethasone has no detectable effect on the apparent rate of degradation of p21 mRNA. Nuclear run-on transcription of the Cip1 gene increases within 2 h after addition of dexamethasone. This effect can be blocked by tetracycline-mediated repression of the glucocorticoid receptor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of fibroblast proliferation is critical for regeneration of wounded tissue. Fibroblast proliferation at wounded sites, as well as other sites in the body, is stimulated by a large number of hormones, including platelet-derived growth factor, fibroblast growth factors, and transforming growth factors. Glucocorticoids, on the other hand, inhibit fibroblast proliferation and delay wound healing. The mechanism of glucocorticoid inhibition of fibroblast proliferation is unclear, and the data on the effects of glucocorticoids on fibroblast proliferation in culture are sometimes conflicting (reviewed in Ref.1). In general, low concentrations of glucocorticoids potentiate the mitotic stimulation that occurs when growth factors are added to serum-starved fibroblasts. On the other hand, higher concentrations of glucocorticoids inhibit proliferation of mid-log phase cultures of fibroblasts.

Naturally occurring and synthetic glucocorticoids inhibit proliferation of mouse L929 fibroblasts in culture (2, 3, 4, 5). Addition of 10-7 M dexamethasone to mid-log L929 culture causes an accumulation of cells with a G1 DNA content (5). Similar results have been observed in glucocorticoid-treated lymphoid cells (6). Inhibition of G1 progression in lymphoid cells is due to the effects of glucocorticoids upon G1 cyclin gene expression (7, 8, 9), and we have undertaken to test the hypothesis that a similar mechanism prevails in fibroblasts.

Progression through the G1 phase of the cell cycle is regulated by a family of serine/threonine protein kinases called cyclin-dependent kinases or Cdks, which are activated by association with a stimulatory subunit, a cyclin (reviewed in Refs. 10–13). Two families or classes of cyclins interact with at least three different Cdks during G1 progression: members of the D cyclin family (cyclin D1, cyclin D2, and cyclin D3) bind to and activate Cdk4 and Cdk6, whereas cyclin E activates Cdk2.

Inhibition of Cdk activity is mediated, in part, by a second set of regulatory subunits, the cyclin kinase inhibitors (reviewed in Refs. 14–16) There are two distinct classes of cyclin kinase inhibitors. Relatives of INK4 bind to Cdk4- and Cdk6-containing complexes, causing dissociation of the cyclin and inhibition of kinase activity (17, 18). One of these inhibitors, p15INK4B, is thought to play a role in TGFß inhibition of keratinocyte proliferation (19, 20). A second family of cyclin kinase inhibitors is comprised of relatives of p21Cip1. Members of this family bind to Cdk2-, Cdk4-, and Cdk6-containing complexes, prevent their activation at low stoichiometry, and block kinase activity at high stoichiometry (21, 22, 23, 24). Induction of p21Cip1 is involved in p53-mediated cell cycle arrest (25), in proliferative senescence (26), in replication and repair of damaged DNA (27, 28), and during differentiation of specific tissue types (29, 30, 31). A relative of p21Cip1 called p27Kip1 is thought to be involved in contact inhibition (32), cAMP inhibition of cell proliferation (33), and TGFß inhibition of epithelial cell proliferation (20, 32, 34, 35).

It is believed that cyclin kinase inhibitors block cell proliferation by virtue of their ability to inhibit Cdk-dependent phosphorylation of critical substrates in the G1 phase of the cell cycle (reviewed in Refs. 14–16). Both Cdk2 and Cdk4 can phosphorylate tumor suppressor gene products, including the product of the Rb-1 gene, pRb, and the related protein p107 (36–39). In cells that express wild type pRb, phosphorylation of pRb is necessary for initiation of S phase (40, 41, 42), and cells that harbor Rb-1 mutations do not require cyclin D for G1 progression (43, 44, 45, 46). The preponderance of evidence indicates that phosphorylation of tumor suppressor gene products by cyclin D/Cdk4 may be central to the role of these kinases in regulating cell cycle progression. Cyclin E/Cdk2 kinase probably plays a role in activating E2F-like transcription factors which, in turn, control nucleotide metabolism in late G1 and S phase (reviewed in Refs. 47 and 48).

The D type cyclins, in conjunction with Cdk4 or Cdk6, appear to be primary targets for hormones that stimulate cell proliferation. D type cyclins are induced by a large number of growth factors (49, 50, 51), and abrogation of D cyclin induction blocks the mitogenic effects of such hormones (52, 53). Hormones that inhibit cell proliferation, such as TGFß, cause a decrease in the amount or activity of cyclin D/Cdk4 complexes (reviewed in Ref.54). Glucocorticoid inhibition of lymphoid cell proliferation is due, in part, to inhibition of cyclin D3 expression (7, 8). We speculated that a similar mechanism might account for glucocorticoid inhibition of fibroblast proliferation.

The experiments described below were undertaken to test the hypothesis that glucocorticoids cause a decrease in the amount or activity of cyclin D/Cdk4 kinase complexes in fibroblasts. We have assayed pRb phosphorylation in vivo and in vitro. pRb kinase activity has been correlated with expression of the catalytic and regulatory subunits of cyclin D1- and D3-dependent kinases. In addition, we have assayed for the expression of two well characterized cyclin kinase inhibitors, p21Cip1 and p27Kip1. The data indicate that glucocorticoids regulate cyclin D/Cdk4 kinase activity in fibroblasts. However, the mechanism is fundamentally different from that described for glucocorticoid inhibition of lymphoid cell proliferation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoid Inhibition of pRb Phosphorylation
Hyperphosphorylation of pRb is associated with, and may be necessary for, G1 progression and initiation of S phase (40, 41, 42). A large number of phosphate residues are attached to pRb, resulting in a significant degree of electrophoretic polymorphism (40, 41, 42). Consequently, the extent of phosphorylation can be assayed by Western blotting. As shown in Fig. 1Go, pRb from mid-log phase L929 cells resolved as a number of species with different electrophoretic mobilities. pRb from dexamethasone-treated cells was much less polymorphic and tended to migrate more rapidly than some of the pRb species that were detected in extracts from untreated mid-log phase cells. These properties are consistent with the conclusion that the state of pRb phosphorylation is reduced in glucocorticoid-treated L929 cells.



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Figure 1. pRb Phosphorylation in Vivo in L929 Cells

Mid-log phase L929 cells were treated with ethanol (control) or dexamethasone (10-7 M). Cell extracts were collected after 24 h and analyzed for the state of pRb phosphorylation by Western blotting.

 
Glucocorticoid inhibition of pRb kinase activity could be demonstrated in vitro. Extracts from control and dexamethasone-treated L929 cells were immunoprecipitated with antibodies against cyclin D1 and cyclin D3 so as to precipitate cyclin D/Cdk4 complexes. These complexes were then assayed for their ability to phosphorylate recombinant glutathione-S-transferase-Rb(GST-Rb) in vitro, as shown in Fig. 2Go. Both cyclin D1- and cyclin D3-associated pRb kinase activity was inhibited by more than 80% after addition of dexamethasone to mid-log phase L929 cells.



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Figure 2. Cyclin D1- and Cyclin D3-Associated pRb Kinase Activity in Vitro

Control and dexamethasone-treated L929 cell extracts were immunoprecipitated with antibodies against cyclin D1 or D3 so as to precipitate the cyclin/Cdk complexes. These complexes were assayed for their ability to phosphorylate recombinant GST-Rb in vitro. The reaction mixture was resolved on polyacrylamide gel and exposed to x-ray film.

 
Glucocorticoids’ Effects on Expression of G1 Cyclins and Cdks
Phosphorylation of pRb is catalyzed during early to mid-G1 by a complex consisting of one of the D-type cyclins and either Cdk4 or Cdk6. Glucocorticoids inhibit D3 cyclin and Cdk4 expression in lymphoid cells (7, 9), and a corresponding decrease in pRb kinase is observed in dexamethasone-treated lymphoid cells (8). Such observations suggest that glucocorticoid inhibition of cyclin D1- and cyclin D3-associated pRb kinase in L929 cells could be due to down-regulation of the regulatory or the catalytic subunit of the G1 pRb kinase (i.e. cyclin D or Cdk4). To test this hypothesis, D cyclins and G1 Cdks were assayed in extracts from control and glucocorticoid-treated L929 cells. As shown in Fig. 3AGo, dexamethasone had no effect on the abundance of Cdk4, Cdk6, cyclin D1, cyclin D2, or cyclin D3. These data are inconsistent with the hypothesis that prolongation of G1 phase in glucocorticoid-treated cells is due to inhibition of D cyclin or Cdk expression. In this respect fibroblasts are very different from lymphoid cells (7, 8, 9).



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Figure 3. Abundance of G1 Cyclins and Cdks and Cyclin/Cdk Complexes in Glucocorticoid-Treated Cells

In the experiment shown in panel A, control and dexa-methasone-treated L929.06 cells were harvested 24 h after treatment. The extracts were assayed for abundance of cyclin D1, cyclin D2, cyclin D3, Cdk4, and Cdk6 by Western blotting. Panel B, Mid-log L929.06 cells were treated with dexamethasone, and extracts were prepared 0, 6, and 24 h after treatment. Cell extracts were immunoprecipitated with antibodies against cyclin D1 or cyclin D3. Immunoprecipitated complexes were dissolved, resolved by electrophoresis, and assayed for Cdk4 by Western blotting.

 
Although glucocorticoids do not affect the abundance of G1 cyclins or Cdks, they might directly or indirectly affect the ability of the D cyclins to bind to their respective Cdks and thereby inhibit the formation of active cyclin D-dependent kinase complexes. To test this hypothesis, control and glucocorticoid-treated L929 cell extracts were immunoprecipitated with antibodies against cyclin D1 and D3. The immunoprecipitates were used to assay Cdk4 to quantify the amount of cyclin D/Cdk complexes in glucocorticoid-treated cells. As shown in Fig. 3BGo, there was no decrease in the amount of Cdk4 that could be coimmunoprecipitated with cyclin D1 or D3. This observation indicates that glucocorticoids do not inhibit the association of D cyclins with Cdk4 in L929 cells.

Glucocorticoid Effects on Cyclin Kinase Inhibitors p21Cip1 and p27Kip1
Cyclin D1- and D3-associated pRb kinase activity is decreased with no corresponding decrease in the abundance of either catalytic or regulatory subunits in glucocorticoid-treated L929 cells. This observation suggests that glucocorticoids may cause an increase in the amount or activity of cyclin kinase inhibitors, such as p21Cip1 or p27Kip1. The abundance of these two inhibitors was measured in control and dexamethasone-treated L929 cells. As shown in Fig. 4Go, the abundance of p27 was relatively high in mid-log phase L929 fibroblasts, and addition of glucocorticoids had no significant effect upon p27 abundance. Unlike p27, the abundance of p21 was very low in mid-log phase L929 cells (Fig. 4Go). Furthermore, dexamethasone induced p21 expression. The abundance of this inhibitor increased by 5- to 10-fold within 6 h and remained high as long as cells were exposed to the steroid.



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Figure 4. Abundance of Cyclin Kinase Inhibitors p21 and p27 in Glucocorticoid-Treated Cells

Mid-log L929.06 cells were treated with dexamethasone. Extracts were collected 0, 6, and 24 h and analyzed for cyclin kinase inhibitors p21 and p27 by Western blot analysis.

 
p21Cip1 Binding to Cdk4 Complexes and Effects on Rb Kinase Activity
The observed increase in the abundance of p21 in glucocorticoid-treated L929 cells suggests the possibility that this kinase inhibitor might bind to G1 cyclin/Cdk complexes and block the kinase activity in L929 fibroblasts. To test whether this increase in the abundance of p21 resulted in an increase in the abundance of p21/Cdk4 complexes, control and glucocorticoid-treated L929 cell extracts were immunoprecipitated with a polyclonal antibody against p21. The abundance of Cdk4 in these immunoprecipitates was assayed by Western blotting. Binding of p21 to Cdk4 increased rapidly, as p21 was induced in glucocorticoid-treated cells. As shown in Fig. 5AGo, the abundance of p21/Cdk4 complexes increased 5- to 10-fold within 6 h and remained constant thereafter.



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Figure 5. Effect of Glucocorticoids on Cdk4-Associated p21 and Rb Kinase Activity

In the experiment shown in panel A, extracts were prepared from cells treated with dexamethasone for 0, 6, and 24 h. Proteins were immunoprecipitated with antibody against p21. The immunoprecipitated complexes were analyzed for the presence of Cdk4 by Western blotting. Panel B, Extracts used in the experiment shown in panel A immunoprecipitated with antibody against Cdk4. The complexes were then assayed for their ability to phosphorylate recombinant GST-Rb in vitro.

 
In parallel experiments, the same extracts that were used in the experiment shown in Fig. 5AGo were immunoprecipitated with a polyclonal antibody against Cdk4. These immunoprecipitates were assayed for their ability to phosphorylate GST-Rb in vitro. As shown in Fig. 5BGo, the kinase activity associated with Cdk4 decreased significantly within 6 h after addition of dexamethasone and remained low thereafter.

Glucocorticoid Induction of p21Cip1 mRNA
In an effort to understand the mechanism whereby glucocorticoids induce p21 expression, RNA was isolated from dexamethasone-treated L929 cells, and the abundance of p21 mRNA was ascertained as shown in Fig. 6AGo. The abundance of p21 mRNA increased 4- to 6-fold within 2 h after addition of dexamethasone. No such induction was observed when dexamethasone was added to E82.A3 cells (Fig. 6BGo), a variant of L929 that is null for the glucocorticoid receptor (55). The expression of p21 mRNA was analyzed in a derivative of E82.A3 that was engineered to expresses the mouse glucocorticoid receptor from a stably integrated, tetracycline-repressible expression vector. This cell line is called E82T4/GR3. As shown in Fig. 6CGo, p21 mRNA was rapidly induced when dexamethasone was added to E82T4/GR3 cells that had been maintained in the absence of tetracycline, so as to de-repress expression of the glucocorticoid receptor. However, no such induction obtained when dexamethasone was added to cells that had been maintained in the presence of 0.1 µM tetracycline for 3 days before addition of the steroid.



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Figure 6. Glucocorticoid Induction of p21 mRNA

In the experiment shown in panel A, mid-log phase L929.06 cultures were treated with dexamethasone, and mRNA was extracted at the intervals indicated. Twenty micrograms of total RNA from each sample were resolved by electrophoresis and quantified by hybridization to labeled probes corresponding to mouse Cip1 cDNA or mouse 18S rRNA. A similar experiment was repeated in panel B using E82.A3 cells, which are null for the glucocorticoid receptor. E82T4/GR3 cells were analyzed in the experiment shown in panel C. Parallel cultures were maintained in the presence or absence of 0.1 µg/ml tetracycline. The cells were treated with dexamethasone and analyzed for p21 mRNA abundance, as shown.

 
The abundance of p21 mRNA reproducibly decreased to near basal expression within 24 h after addition of dexamethasone to wild type L929 cells, as shown in Fig. 6AGo. On the other hand, glucocorticoid induction of p21 mRNA was more robust and more persistent in E82T4/GR3 cells, when such cells were exposed to dexamethasone in the absence of tetracycline (Fig. 6CGo). Glucocorticoid-mediated down-regulation of the glucocorticoid receptor is characteristic of L929 cells (56) and might account for the transitory pattern of p21 mRNA induction. Figure 7Go shows Western blotting data in which the glucocorticoid receptor was measured in those L929 variants that were analyzed for p21 induction. The first four lanes contain the results from the tetracycline-repressible E82T4/GR3 cells, and lanes 1 and 3 reveal the extent to which the glucocorticoid receptor is de-repressed by withdrawal of tetracycline. The abundance of the receptor in the fully de-repressed E82T4/GR3 cells (lane 3) was at least 5 times greater than that observed in wild type L929 cells (lane 5). The expression of the glucocorticoid receptor decreased after addition of dexamethasone to both wild type (lane 6) and E82T4/GR3 cells (lane 4), but the abundance of the receptor in the E82T4/GR3 cells 24 h after dexamethasone (lane 4) was equal to or greater than that observed in control L929 cells (lane 5). This apparent overexpression of the glucocorticoid receptor may account for the extent to which the induction of p21 mRNA persists after addition of dexamethasone to E82T4/GR3 cells (as shown in Fig. 6CGo).



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Figure 7. Regulation of Glucocorticoid Receptor Expression in L Cell Lines

Extracts were prepared from mid-log phase cells or from cultures that had been treated for 24 h with dexamethasone. In addition, cultures were prepared from E82T4/GR3 cells that had been maintained in the presence of 0.1 µg/ml tetracycline (lanes 1 and 2). Forty micrograms of total cellular protein were resolved by electrophoresis, blotted to nitrocellulose filters, and probed with BUGR anti-glucocorticoid receptor antibody, as described in Materials and Methods. The predicted position of the 94-kDa glucocorticoid receptor is indicated by the arrow.

 
Induction of Cip1 Transcription in Glucocorticoid-Treated Cells
The data shown in Figs. 6Go and 7Go indicate that induction of p21 expression in L929 cells depends upon expression of the glucocorticoid receptor. Furthermore, induction of p21 mRNA was not blocked when cells were exposed to cycloheximide for 2 h before addition of dexamethasone, as shown in Fig. 8AGo. Neither did dexamethasone alter the apparent rate of degradation of p21 mRNA, as shown in Fig. 8BGo. In this experiment, the abundance of p21 mRNA was measured at intervals after addition of actinomycin D to control L929 cells and to cells that had been treated with dexamethasone for 2 h before addition of actinomycin D. The apparent half-life of p21 mRNA was 4–6 h, irrespective of the presence of glucocorticoids. The observation that p21mRNA stability is not altered by dexamethasone suggests that glucocorticoids must increase transcription of Cip1; moreover, the observation that induction of p21 mRNA cannot be blocked by cycloheximide suggests that such induction may be due to a direct interaction between the Cip1 promoter and the glucocorticoid receptor. Nuclear run-on transcription was carried out to determine whether induction of p21 mRNA was associated with increased Cip1 transcription, as shown in Fig. 8CGo. Cip1 transcription was increased after addition of dexamethasone to E82T4/GR3 cells. When the data from three independent experiments were quantified and normalized to transcription of the 5S RNA gene (as shown in the bar graph in Fig. 8CGo), the mean stimulation was estimated to be 4- to 6-fold, consistent with the increase in p21 mRNA that one observes within 2 h after addition of dexamethasone. The glucocorticoid-mediated increase in Cip1 transcription was blocked when cells were grown in the presence of tetracycline, so as to repress the glucocorticoid receptor, before addition of dexamethasone (Fig. 8CGo).



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Figure 8. Effects of Cycloheximide and Actinomycin D on p21 mRNA Expression

In the experiment shown in panel A, mid-log phase cultures were treated for 2 h with 5 µg/ml cycloheximide, as indicated. Dexamethasone was added and RNA was extracted after 2 h in the presence of steroid. The abundance of p21 mRNA was assessed by Northern blotting, using 20 µg total cellular RNA. In the experiment shown in panel B, parallel cultures were prepared. One was treated for 2 h with dexamethasone (•) while the other was untreated ({circ}). Actinomycin D was added to a final concentration of 1 µg/ml, and RNA was extracted at intervals thereafter. The abundance of p21 mRNA was determined by Northern blotting, and the data were quantified by densitometry. Curves depicting decay kinetics were fitted by first-order linear regression analysis.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The product of the retinoblastoma susceptibility gene, pRb, is the prototypic tumor suppressor protein. Although the mechanism of action of pRb is far from understood, there are certain general features of this regulatory entity that may be perceived. It would appear that pRb blocks progression through the G1 phase of the cell cycle. This blockade is relieved by extensive phosphorylation, which occurs during mid- to late G1 phase (37–42, 57–59). Available data are consistent with the hypothesis that phosphorylation of pRb releases an array of proteins including, but probably not limited to, transcription factors such as E2F-1 and UBF-1 (47, 60). These factors then stimulate or sustain an ordered reaction pathway that culminates in the cellular commitment to initiate DNA replication. When cells fail to phosphorylate pRb, they will arrest in G0/G1 phase (48).

Several enzymes phosphorylate pRb. Cdk4 and Cdk6 phosphorylate pRb and the related pocket protein p107 (61, 62), and there are data that suggest that these enzymes have no other essential function (43, 44, 45, 46). Cdk2 can also phosphorylate pRb (63, 64, 65, 66), but there is controversy concerning the extent to which Cdk2-dependent phosphorylation of pRb is essential to cell cycle progression (67). Despite an incomplete understanding of the enzymology, it is clear that hormonal regulation of cell proliferation impinges upon pRb phosphorylation. A wide variety of growth-promoting hormones regulate the expression of one or another of the cyclin subunits that are required for activation of the pRb kinases. This type of regulation was first shown for CSF-1, which is necessary for expression of cyclin D1 in myelogenous cells (49). Virtually every growth-promoting hormone that has been examined to date stimulates the expression of one of the D-type cyclins (61, 68). The data are consistent with the proposition that members of the D cyclin family are among the primary targets for all mitogenic hormones.

Phosphorylation of pRb is also regulated by hormones that inhibit cell proliferation. TGFß inhibits pRb phosphorylation and causes G1 arrest in sensitive epithelial cells (69), whereas glucocorticoids inhibit pRb phosphorylation and cause G1 arrest in murine lymphoma cells (8). Both hormones cause a decrease in the amount or the activity of a cyclin D/Cdk4 (or cyclin D/Cdk6) pRb kinase, although the effects of TGFß depend to an extent upon the cell line that is studied (19, 20, 54, 70). The mechanism of inhibition of pRb phosphorylation is fairly straightforward in glucocorticoid-arrested T lymphoma cells. Glucocorticoids inhibit the expression of the major D-type cyclin in cells of thymic origin, i.e. cyclin D3 (7). The activity of Cdk4 (and probably Cdk6) decreases in consequence and the cells arrest in G1 (9). Antiestrogens inhibit expression of cyclin D1 in breast cancer cells (71), and there are data that suggest that glucocorticoid inhibition of Con8 breast cancer cell proliferation may involve changes in cyclin D1 expression (72). Taken together, these data suggest that steroid hormones that inhibit cell proliferation may do so by virtue of their ability to down-regulate one of the members of the D cyclin family.

Glucocorticoid inhibition of fibroblast proliferation appears to involve a different mechanistic strategy. Glucocorticoids inhibit pRb phosphorylation, and everything that we know about cell cycle progression indicates that this effect is sufficient to block entry into S phase. Moreover, inhibition of pRb phosphorylation is due to a glucocorticoid-mediated decrease in the activity of cyclin D/Cdk4. Thus, glucocorticoids regulate the proliferation of both lymphoid and fibroblastic cells by a common device: inhibition of cyclin D/Cdk4 pRb kinase activity. Unlike the situation that obtains in lymphoid cells, one does not observe any significant change in the expression of any of the D cyclins or their catalytic partners, Cdk4 or Cdk6, in glucocorticoid-treated L929 cells. Inhibition of cyclin D/Cdk4 kinase activity in the absence of any change in the expression of either the cyclin or Cdk subunit strongly suggests that a cyclin kinase inhibitor is involved in the response. The effects of dexamethasone appear to be mediated by induction of p21Cip1, which increases 5- to 10-fold after addition of glucocorticoids to mid-log phase cells. There is a corresponding increase in the abundance of ternary complexes that contain Cdk4 and p21, and these ternary complexes either lack or have very little Cdk4 kinase activity.

Expression of the Cip1 locus is rapidly induced in glucocorticoid-treated cells. The abundance of p21 mRNA increases >5 fold within 2 h after addition of dexamethasone. In wild type L929 cells, the glucocorticoid receptor is down-regulated after addition of dexamethasone, and one observes that p21 mRNA expression begins to decrease about 12 h after addition of the steroid. Nevertheless, the abundance of p21 protein remains relatively constant for at least 24 h, suggesting that several mechanisms probably impinge on p21 expression in glucocorticoid-treated cells. Induction of p21 mRNA is associated with an increase in nuclear run-on transcription of Cip1. We have been unable to detect any change in p21 mRNA degradation in glucocorticoid-treated cells, although one must be cautious when interpreting mRNA degradation data obtained by the use of nonspecific inhibitors such as actinomycin D. The simplest explanation of the data is that glucocorticoids activate transcription of Cip1, and p21 mRNA increases thereafter. The effect is totally dependent upon expression of functional glucocorticoid receptors, and induction of p21 mRNA can be demonstrated in the presence of nonspecific inhibitors of translation. The rapidity of the response, the requirement for glucocorticoid receptor, and the insensitivity to cycloheximide argue that activation of Cip1 is due to a direct or primary interaction between the Cip1 promoter and the glucocorticoid receptor. Experiments are currently in progress to identify glucocorticoid response elements within the Cip1 promoter, but preliminary data indicate that no such element exists within 3.4 kb upstream of the transcriptional start site.

The data presented above indicate that glucocorticoids induce transcription of Cip1. The consequent increase in p21 protein abundance results in a decrease in cyclin D/Cdk4 kinase activity and a concomitant inhibition of pRb phosphorylation. Inhibition of pRb phosphorylation accounts, at least in part, for glucocorticoid inhibition of fibroblast proliferation. It is interesting to note that glucocorticoids do not cause G0 arrest in L929 cells. Instead, G1 progression is attenuated at some point in mid- to late G1 (5, 73). Cells escape from this pause point, enter S phase, and complete cell division with a frequency that is sufficient for a population-doubling time of >100 h. If one assumes that a certain degree of Rb phosphorylation much be achieved before S phase can be initiated in normal fibroblasts, then one must assume that phosphorylation of pRb increases slowly at this pause site, to the extent that the protein ultimately accumulates the appropriate number of types of phosphorylations to permit G1 progression and entry into S phase. In order for such phosphorylation events to accrue slowly, the rate of dephosphorylation of pRb during G1 must be very low, so that even very low levels of pRb kinase activity can, given sufficient time, ultimately achieve critical phosphorylation of the tumor suppressor protein. The biochemistry of pRb phosphorylation is very complex and poorly understood, and less is known about dephosphorylation of these key regulators of G1 progression. It is clear, however, that these processes will prove to be central to hormonal control of cell division, and our understanding of how hormones regulate the commitment to DNA replication must ultimately be understood in terms of the phosphorylation state of pRb.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
L929.06 cell line is a subclone of the mouse L cell (NCTC 929) selected for its sensitivity to dexamethasone (71). The L cell clone designated E82.A3 is a null variant isolated and characterized in Housley’s laboratory (55). W. V. Vedeckis’ laboratory engineered E82.A3 to express the mouse glucocorticoid receptor from a stably integrated tetracycline repressible expression vector, and a detailed description of these cells will be published elsewhere (P. Wei, Y. I. Ahn, P. R. Housley, J. Alam, and W. V. Vedeckis, manuscript submitted). L cells were grown in DMEM supplemented with 5% FBS and subcultured by treatment with trypsin.

Reagents
The anti-p21 antibody used in Western blotting was obtained from Dr. David Hill of Oncogene Science Inc. (Cambridge, MA). All other antibodies (including anti-p21 antibody used for immunoprecipitation) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Recombinant GST-Rb protein was prepared from Escherichia coli extracts, as previously described (8).

Western Blotting and Immunoprecipitation
Immunoblotting was carried out essentially as described by Rhee et al. (9). Briefly, cells were harvested by trypsinisation, collected by centrifugation (at 4 C), and lysed for 15 min on ice in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, with 10 µg/ml each of aprotinin, phenylmethylsulfonyl fluoride, leupeptin, and pepstatin added just before use). Cell lysates were cleared by centrifugation (in a microfuge at maximum speed for 15 min at 4 C), and protein concentration was determined by the method of Bradford (74). Samples were denatured by boiling for 5 min in 2x sample buffer containing 0.125 M Tris-HCl, pH 6.8, 1% SDS, 20% glycerol, and 10% ß-mercaptoethanol. Samples (60 µg/lane) were separated by SDS-PAGE on 12% polyacrylamide gels using a mini gel apparatus (Bio-Rad, Richmond, CA). Proteins were then transferred onto nitrocellulose membrane (Schleicher & Schuell, Keene, NH) using a semidry transfer apparatus (Bio-Rad). Membranes were incubated overnight at room temperature in blocking solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20, 5% nonfat dry milk), followed by a 3-h incubation at room temperature in primary antibody diluted in blocking solution. Membranes were washed three times in blocking solution, followed by a 1-h incubation in horseradish peroxide-conjugated secondary antibody diluted in blocking solution. Membranes were washed three times in blocking solution before the specific protein bands were detected by the enhanced chemiluminescence system (Amersham, Arlington Heights, IL).

Immunoprecipitation was undertaken by the same method as above except that before separation of the proteins by SDS-PAGE, equal amounts (150 µg) of protein were incubated with indicated antibody for 2 h on ice with occasional mixing. Antigen-antibody complexes were recovered by incubation for 2 h at 4 C with 30 ml Protein A-Sepharose beads (0.1 g suspended in 1 ml lysis buffer) (Sigma, St. Louis, MO), washed four times in lysis buffer, and dissolved in 2x sample buffer followed by separation and detection as described above.

Rb Kinase Assay
Phosphorylation of recombinant GST-Rb was assayed as described by Rhee et al. (8). Approximately 105 cells were washed in PBS and lysed by adding 100 µl IP buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM ß-glycerophosphate, 1 mM NaF, 10% glycerol, 0.1 mM sodium orthovanadate, 1 mM dithiothreitol (DTT), plus 10 µg each of aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride added freshly), followed by sonication of three 15-sec bursts with cooling on ice in between. The sonicated mixtures were clarified by centrifugation at 12,000 x g at 4 C. The protein content of the supernatant fraction was measured, and 500 µg of protein were transferred to a microcentrifuge tube along with 1 µl of cyclin D or Cdk4 polyclonal antibody. This mixture was incubated for 1–2 h on ice with occasional mixing. Fifty microliters of protein A-Sepharose beads were added to the mixture and incubated for 2–3 h at 4 C with rocking. The beads were sedimented by centrifugation for 30 sec at 4 C at 10,000 x g and washed three times with IP buffer and twice with 50 mM HEPES, pH 7.5, containing 1 mM DTT. Twenty two microliters of kinase buffer containing 1.5 µg recombinant GST-Rb, 10 mCi [{alpha}-32P]ATP (6000 Ci/mmol), 50 mM HEPES, pH 7.5, 10 mM MgCl2, 2.5 mM EGTA, 10 mM ß-glycerophosphate, 1 mM sodium fluoride, 1 mM DTT, and 0.1 mM sodium orthovanadate were added to the beads. The reaction mixture was incubated at 30 C for 30 min. The reaction was stopped by adding 25 µl of 2x SDS-PAGE sample buffer, and the samples were processed for electrophoresis on a 6% polyacrylamide gel. The gel was dried onto a Whatman 3MM paper (Clifton, NJ) and autoradiography carried out.

Cip1 Gene Expression
Analysis of p21 mRNA abundance was carried out by Northern blotting (5, 6, 7). Mouse Cip1 cDNA was labeled and used as a probe. Nuclear run-on transcription was assayed using published procedures (5, 6, 7). Mouse Cip1 cDNA was used to measure Cip1 transcription, and transcription of 5S RNA was measured as an internal control. All autoradiographic and chemiluminescent data were analyzed using a Lynx 5000 digital image analysis system with version 5.5 software. Exposures were controlled so as to provide a linear response range of at least 5-fold.



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Figure 9. Nuclear Run-on Transcription of Cip1 in Glucocorticoid-Treated Cells

Mid-log phase cultures of E92T4/GR3 cells were grown in the absence or presence of 0.1 µg/ml tetracycline. Cultures were treated with dexamethasone for 2 h, and parallel cultures were untreated. Nuclei were isolated and transcription was carried out in the presence of [{alpha}32P]UTP. Labeled RNA was extracted and hybridized to Cip1 cDNA and 5S RNA genes immobilized on nitrocellulose filters, as described in Materials and Methods. Hybridization was measured by densitometry, and data were normalized to transcription of 5S RNA genes.

 

    ACKNOWLEDGMENTS
 
The authors wish to express particular thanks to Ping Wei and Wayne Vedeckis for providing the tetracycline-repressible GR expression clone E82T4/GR3 and to Wade Harper for providing mouse Cip1 cDNA.


    FOOTNOTES
 
Address requests for reprints to: E. Aubrey Thompson, Ph.D., Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-0645.

This work was supported in part by Grant P01-AG10514 from the National Institute of Aging and Grant R37-CA24347 from the National Cancer Institute.

Received for publication April 15, 1996. Revision received January 30, 1997. Accepted for publication February 5, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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