Ubiquitination and Proteasome-Mediated Degradation of BRCA1 and BARD1 during Steroidogenesis in Human Ovarian Granulosa Cells

Abstract

Germ-line mutations in BRCA1 predispose women to early-onset, familial breast and ovarian cancers. However, BRCA1 expression is not restricted to breast and ovarian epithelial cells. For example, ovarian BRCA1 expression is enriched in ovarian granulosa cells, which are responsible for ovarian estrogen production in premenopausal women. Furthermore, recent tissue culture and animal studies suggest a functional role of BRCA1 in ovarian granulosa cells. Although levels of BRCA1 are known to fluctuate significantly during folliculogenesis and steroidogenesis, the mechanism by which BRCA1 expression is regulated in granulosa cells remains to be elucidated. Here we show that the ubiquitin-proteasome degradation pathway plays a significant role in the coordinated protein stability of BRCA1 and its partner BARD1 in ovarian granulosa cells. Our work identifies the amino-terminal RING domain-containing region of BRCA1 as the degron sequence that is both necessary and sufficient for polyubiquitination and proteasome-mediated protein degradation. Interestingly, mutations in the RING domain that abolish the ubiquitin E3 ligase activity of BRCA1 do not affect its own ubiquitination or degradation in ovarian granulosa cells. The proteasome-mediated degradation of BRCA1 and BARD1 also occurs during the cAMP-dependent steroidogenic process. Thus, the dynamic changes of BRCA1/BARD1 protein stability in ovarian granulosa cells provide an excellent paradigm for investigating the regulation of this protein complex under physiological conditions.

GERM-LINE MUTATIONS IN BRCA1 account for a significant percentage of familial breast and ovarian cancers (1, 2). A large body of evidence has implicated BRCA1 in multiple essential nuclear functions including DNA repair, DNA damage checkpoint, gene regulation, centrosome duplication, etc. (1, 3, 4). At the biochemical level, the N-terminal RING domain of BRCA1 interacts with another RING domain-containing protein BARD1, and the BRCA1/BARD1 heterodimeric complex confers strong ubiquitin E3 ligase activity (5, 6). A compromised function of BRCA1 in the maintenance of genome stability most likely contributes to cancer development. However, because BRCA1 expression is not restricted to breast and ovarian epithelial cells, it remains a conundrum as to how loss of the universally important DNA repair function of BRCA1 would lead to tissue- and gender-specific cancers.

Estrogen is known to play a paramount role in the development of estrogen receptor α (ERα)-positive breast cancer (7–9). Although BRCA1-associated tumors tend to be basal like and ERα negative (1, 10, 11), prophylactic oophorectomy significantly reduces the risk of both breast and ovarian cancers in carriers of BRCA1 mutations (12, 13). This suggests that, as it does to sporadic breast cancers, circulating estrogen in premenopausal women may also contribute to the initiation of BRCA1-associated tumors. The ability of BRCA1 to directly bind to ERα and inhibit its transcription activity in breast cancer cells represents one potential mechanism by which BRCA1 confers its tumor suppressor function in the major estrogen-responsive tissues (14–17).

Historically, functional studies of BRCA1 have been carried out predominantly in breast epithelial/carcinoma cells. However, BRCA1 expression is not limited to breast epithelial cells (18, 19), and BRCA1 could conceivably play important biological functions in nonepithelial cells as well. Notably, expression of mouse ovarian Brca1 is highly enriched in granulosa cells of developing follicles and appears to fluctuate during folliculogenesis (19–21). In preovulatory follicles, FSH triggers a surge of intracellular cAMP level. This, in turn, stimulates expression of aromatase, a key enzyme in estrogen biosynthesis in both ovarian granulosa cells and certain extragonadal tissues such as adipose tissue (22). Concomitant with increased aromatase expression upon cAMP stimulation in vitro, the BRCA1 protein levels in ovarian granulosa cells are significantly reduced (23). A similar inverse correlation of BRCA1 and aromatase expression was also observed in adipose stromal cells (24, 25). Importantly, small interfering RNA-mediated reduction of BRCA1/BARD1 expression in human ovarian granulosa cells and adipose stromal cells stimulates aromatase expression (23–25). In addition, tissue-specific knockout (KO) of Brca1 in mouse ovarian granulosa cells induces ovarian and uterine tumors that still carry wild-type alleles of Brca1 (26). Taken together, these findings raise the distinct possibility that loss of BRCA1 in steroidogenic nonepithelial cells may result in aberrant estrogen production, which could increase the risk of tumor formation in estrogen-responsive tissues.

In light of the dynamic change of BRCA1 levels and its potential physiological role in ovarian granulosa cells, we investigated the underlying mechanism by which levels of BRCA1 and its partner BARD1 are regulated in this particular cellular context. Our work indicates that the ubiquitin/proteasome-mediated protein degradation pathway modulates the BRCA1/BARD1 protein stability in ovarian granulosa cells. Our study also suggests that the N-terminal region of BRCA1 and multiple lysine residues within it mediate the polyubiquitination and proteasome actions.

RESULTS

Expression of Brca1 and Bard1 in Cumulus and Mural Granulosa Cells in Mouse Ovaries

Although a functional antagonism between BRCA1/BARD1 and aromatase expression has been suggested by in vitro tissue culture studies (23–25), there lacks a direct comparison of the expression of all three genes in ovarian follicles. FSH-stimulated aromatase expression predominantly occurs in mural granulosa cells, which are associated with the basal lamina (27). In contrast, cumulus granulosa cells, which are closely associated with the oocyte, do not express abundant aromatase (28). To compare the levels of Brca1, Bard1, and aromatase in mouse ovaries, mural granulosa cells (Mural) and cumulus-oocyte complex (COC) fractions were prepared from large antral and preovulatory follicles as described in detail in Materials and Methods. As shown in Fig. 1, A and B, both the mRNA and protein levels of Brca1 and Bard1 were significantly lower in mural granulosa cells than the COC fraction, whereas the opposite pattern was observed for aromatase. The Brca1/Bard1 signals from the COC fraction are most likely contributed by cumulus granulosa cells, because denuded oocytes (oocyte free of cumulus cells) express Brca1 and Bard1 at much lower levels than COC (data not shown). Thus, the expression of both Brca1 and Bard1 is reciprocally correlated with aromatase expression in murine ovarian follicles.

Fig. 1.

Brca1, Bard1, and Aromatase (Cyp19) Expression in Mouse Ovarian Follicular Cells A, Real-time PCR analysis of the expression of Brca1, Bard1, and aromatase in mural granulosa cells (Mural) and COC. 18s rRNA was used for normalization. B, Comparison of Brca1, Bard1, and aromatase protein levels in Mural and COC fractions. α-Tubulin was used as a loading control.

The N-Terminal Region of BRCA1 Confers Proteasome-Mediated Protein Degradation

We next explored the underlying mechanisms by which BRCA1/BARD1 levels are regulated in ovarian granulosa cells. Although both the mRNA and protein levels of Brca1/Bard1 follow the same trend in mural and cumulus granulosa cells (Fig. 1), the difference in protein abundance between the two granulosa cell types seems more dramatic than that in mRNA, suggesting that regulation of translation and/or protein stability may be involved. To facilitate the mechanistic study, we used a human granulosa cell line (KGN) that displays a similar steroidogenic capability as primary granulosa cells (29). As shown in Fig. 2A, both BRCA1 and BARD1 proteins can be stabilized by the MG132 treatment. Other proteasome inhibitors such as ALLN resulted in a similar degree of protein stabilization (data not shown). This result indicates that the proteasome-dependent protein degradation pathway contributes to the stability of BRCA1/BARD1 in ovarian granulosa cells.

Fig. 2.

The N-Terminal Region of BRCA1 Confers Proteasome-Dependent Degradation of the Protein in a Human Ovarian Granulosa Cell Line (KGN) A, KGN cells were treated with either vehicle or proteasome inhibitor MG132 (5 μm) for 16 h. Whole-cell extract was prepared and analyzed for the expression of BRCA1 and BARD1. B, Six overlapping fragments that cover the entire BRCA1 open reading frame are expressed in KGN cells. A leading peptide containing the Flag tag, HA tag, and the NLS derived from SV40 large T antigen was fused with the individual fragment. KGN cells expressing various BRCA1 fragments were treated with MG132 (5 μm) for 16 h. Cells were then harvested for the analysis of the expression of BRCA1 fragments using an α-Flag antibody. The positions of the fragments are indicated by asterisks. C, An expression vector for either native EGFP or EGFP fused with BRCA1 Fragment 1 (1–324 aa) was transfected into KGN cells. The vehicle or MG132-treated samples were analyzed by immunoblotting with an α-EGFP antibody. D, An expression vector for the Flag-tagged full-length or N terminus truncated BRCA1 (Δ1–302 aa) was transfected into HEK293T cells. Transfected cells were incubated overnight and then treated with vehicle or MG132 (5 μm) for 12 h. The ectopically expressed proteins were detected by immunoblotting with an α-Flag antibody. IB, Immunoblotting.

To determine the region of BRCA1 that confers the protein degradation, six overlapping BRCA1 fragments that cover the entire BRCA1 open reading frame were expressed in KGN cells (Fig. 2B). Fragments 2–6 were expressed at a comparable level and none of them was further stabilized by the MG132 treatment (lanes 3–12; Fig. 2B). In contrast, the level of Fragment 1 [amino acids (aa) 1–324] was significantly elevated in the presence of MG132 (lanes 1 and 2; Fig. 2B), suggesting that this fragment was actively degraded by the proteasome machinery. To determine whether the same N-terminal region of BRCA1 (1–324) is sufficient to confer proteasome-mediated protein degradation in a heterologous context, this part of BRCA1 was fused to the enhanced green fluorescent protein (EGFP). In contrast to the abundance of the native EGFP (lanes 1 and 2 in Fig. 2C), EGFP fused with Fragment 1 was substantially stabilized by MG132 (compare lanes 3 and 4 in Fig. 2C). Thus, the 324-aa N-terminal region of BRCA1 can serve as a degron sequence in conferring proteasome-dependent protein degradation.

Given that the full-length BRCA1 can be stabilized by MG132 (30)(Fig. 2A), we asked whether the N-terminal 324 aa could mediate the proteasome-dependent degradation of the full-length BRCA1 protein. Due to the technical difficulty in expressing the full-length BRCA1 in KGN cells, the wild-type and N-terminal truncated BRCA1 proteins were ectopically expressed in human embryonic kidney (HEK)293T cells. As shown in Fig. 2D, the full-length BRCA1 protein was stabilized by the MG132 treatment (lanes 3 and 4), consistent with the findings of the endogenous BRCA1 protein in KGN cells. However, the stabilizing effect of MG132 was not observed with the N-terminal truncated protein (lanes 5 and 6; Fig. 2D). Taken together, our data suggest that the N-terminal region of BRCA1 (1–324) is both necessary and sufficient for mediating the proteasome action.

Multiple Lysine Residues in the BRCA1 N-Terminal Region Can Serve as the Ubiquitination Sites

To further map the region in Fragment 1 that confers proteasome-mediated degradation, we generated two smaller fragments that cover the N- and C-terminal halves of the fragment (1-N: aa 1–167; and 1-C: aa 168–324). As shown in Fig. 3A, 1-N was expressed at a much lower level than 1-C (compare lanes 5 and 7). Furthermore, 1-N, but not 1-C, was substantially stabilized by MG132 (compare lanes 5 and 6), suggesting that the first 167 aa residues of BRCA1 are targeted for proteasome-mediated degradation.

Fig. 3.

Multiple Lysine Residues in the First 167-aa Region of the N-Terminal BRCA1 Are Involved in Ubiquitination and Proteasome-Dependent Degradation A, Fragment 1 was further divided into the amino and carboxyl-terminal halves. Stability of these two fragments in KGN cells was examined after transient transfection and treatment with MG132. Asterisks indicate the bands that correspond to the ubiquitinated species. B, The primary sequence of 1-N is shown with the 14 lysine residues underlined. Stability of the wild-type fragment and the all 14-lysine mutant (14KR) were compared by immunoblotting. To eliminate the contribution of exogenous lysine residues to the ubiquitination and degradation of the BRCA1 fragment, the foreign sequences that encode the tags and NLS were removed from the expression constructs. The proteins were detected with an α-BRCA1 antibody (Ab1). IB, Immunoblotting; (Ub)n, polyubiquitination.

Proteasome-mediated protein degradation is triggered by polyubiquitination whereby multiple ubiquitin molecules are conjugated to lysine residues of the target protein (31–33). Upon blocking of the proteasome activity, polyubiquitinated protein species accumulate and can be readily detected by immunoblotting. Indeed, slower-migrating bands with sizes corresponding to the ubiquitinated species were found in the anti-Flag immunoblotting of MG132-treated samples for Fragments 1 and 1-N (asterisks in Fig. 3A). The protein ladder became even more prominent when an anti-BRCA1 antibody (Ab1) was used for the detection of Fragment 1-N (lane 4 in Fig. 3B).

There are a total of 14 lysine residues within the first 167-aa region of BRCA1 (1-N), all of which are conserved among vertebrate orthologs of BRCA1 (underlined in the aa sequence in Fig. 3B). Change of part, or all, of the 14 lysine residues simultaneously to arginine did not affect the stability or ubiquitination pattern of the Flag-tagged 1-N fragment (data not shown; and supplemental Fig. 1A published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Given the commonly observed promiscuity in the choice of lysine residues for ubiqutination, we suspected that the exogenous lysine residues present in the Flag-containing tag might have served as substitute sites for ubiquitination. Indeed, the 14KR mutant in the absence of any tag sequence was substantially more stable than the untagged wild-type counterpart (compare lanes 3 and 5 in Fig. 3B). Furthermore, the untagged 14KR mutant lost the slower-migrating protein ladder indicative of the ubiquitinated species. Change of a subset of the 14 lysine residues was not sufficient for protein stabilization or removal of the protein ladder (supplemental Fig. 1B). Taken together, our result suggests that the putative ubiquitin E3 ligase can use multiple lysine residues in the BRCA1 fragment as the attachment sites for polyubiquitination.

Polyubiquitination of the N-Terminal BRCA1 by Ectopically Expressed Ubiquitin

To directly demonstrate polyubiquitination of the first 167-aa region of BRCA1, HEK293T cells were cotransfected with the expression vectors for Fragment 1-N and histidine-tagged ubiquitin (His-Ub). His-Ub was precipitated with nickel-agarose beads and immunoblotted with an anti-BRCA1 antibody (Ab1). As shown in Fig. 4, the His-Ub-containing ubiquitinated BRCA1 fragment was detected in the cotransfected cells (lane 9; top panel), and was substantially enriched in the presence of MG132 (lane 10; top panel). Cotransfection with a mutant ubiquitin (KO), in which all lysine residues in ubiquitin were mutated to arginine (34), resulted in a diminished signal for the Ni-NTA-agarose-bound species (lanes 11 and 12; top panel of Fig. 4), consistent with the fact that the KO mutant blocks polyubiquitination. Furthermore, His-Ub was not associated with any appreciable amount of the untagged 14KR mutant (lanes 7–12; bottom panel of Fig. 4), thus unequivocally demonstrating the involvement of these multiple lysine residues in ubiquitination of the BRCA1 N-terminal fragment.

Fig. 4.

Demonstration of Ubiquitination of 1-N Using Ectopically Expressed His-Ub The His-tagged wild type (WT) or mutant ubiquitin (KO) were coexpressed in HEK293T cells with either 1-N-WT or 1-N-14KR. The 1-N proteins conjugated with the His-tagged ubiquitin were pulled down with Ni-NTA agarose beads and visualized by immunoblotting with an α-BRCA1 antibody. IB, Immunoblotting; IP, immunoprecipitation; (Ub)n, polyubiquitination.

The BRCA1/BARD1 Ubiquitin E3 Ligase Activity Is Not Required for BRCA1 Ubiquitination or Degradation in KGN Cells

The BRCA1 RING domain has been reported to catalyze autoubiquitination via the linkage at lysine 6 (K6) of ubiquitin (6, 35–40). Although K6-conjugated ubiquitination is not generally considered to signal proteasome-mediated degradation, it was still possible that the RING domain-mediated autoubiquitination might contribute to the BRCA1 degradation observed in the current study. To test this possibility, we compared the wild-type BRCA1 fragment (aa 1–324) and two well-characterized cancer-predisposing mutants (C61G and G64G) that lack the ubiquitin E3 ligase activity. As shown in Fig. 5A, both mutants were as unstable as their wild-type counterpart, and they were readily stabilized after the treatment with MG132 (lanes 3–8 in the top panel). In addition, the mutants gave rise to the same pattern of polyubiquitin chains as the wild-type protein.

Fig. 5.

BRCA1/BARD1 Does Not Mediate Proteasome-Dependent Degradation of BRCA1 A, Comparison of the wild-type BRCA1 1-N fragment with two cancer-predisposing mutants (C61G and C64G) that lack the ubiquitin E3 ligase activity. B, Ectopically expressed BARD1 enhances the stability of BRCA1 1-N fragment. An expression vector expressing the wild-type BARD1 was cotransfected into KGN cells with either wild-type or mutant BRCA1 Fragment 1 (C61G or C64G). MG132-treated and untreated cells were harvested for protein analysis using antibodies as indicated. IB, Immunoblotting; WT, wild type; (Ub)n, polyubiquitination.

BARD1 forms a stable heterodimeric complex with BRCA1 via the RING domains of both proteins and greatly enhances the ubiquitin E3 ligase activity (5, 6). This raised a possibility that BARD1 might mediate BRCA1 ubiquitination and degradation in KGN cells. However, as shown in Fig. 5B, ectopic expression of BARD1, in fact, led to substantial stabilization of Fragment 1 of BRCA1 (compare lane 3 with 9). The stabilizing effect of BARD1 on BRCA1 was most likely due to the physical association between the two proteins, because the two cancer-predisposing mutations of BRCA1 that disrupt the BRCA1/BARD1 interaction (C61G and C64G) (41) prevented the BARD1-mediated stabilization (compare lane 9 with 11 and 13 in the top panel of Fig. 5B). In addition, a RING domain deletion mutant of BARD1 also impaired the stabilizing effect of BARD1 on BRCA1 (supplemental Fig. 2 published as supplemental data on The Endocrine Society’s Journals Online web site). Therefore, BARD1 is unlikely to be responsible for the ubiquitination and proteasome-dependent degradation of BRCA1 in KGN cells. Rather, a ubiquitin E3 ligase other than BRCA1/BARD1 may mediate the ubiquitin/proteasome-dependent degradation of the BRCA1 in ovarian granulosa cells.

Proteasome-Mediated Degradation of BRCA1/BARD1 in KGN Cells after cAMP Surge

Steroidogenesis in ovarian granulosa cells is strongly induced by elevation of cAMP levels. This process can be recapitulated in vitro by treating KGN cells with activator of adenylyl cyclase forskolin (FSK) or cAMP analog N⁶,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate [(Bu)₂cAMP] (29, 42). Consistent with published findings (42), both the mRNA and protein levels of aromatase were significantly elevated after the treatment of either FSK or (Bu)₂cAMP (top panel in Fig. 6A and open circle in 6B). This was accompanied by gradual reduction of BRCA1 and BARD1 proteins (middle two panels in Fig. 6A). The mRNA levels of BRCA1 and BARD1 were also decreased after the FSK treatment, but were not apparent until 18 h post induction (open diamond and solid square in Fig. 6B). Therefore, the abundance of BRCA1/BARD1 in KGN cells after the cAMP surge may be regulated at both mRNA and protein levels. In support of this notion, MG132 treatment prevented the FSK-induced degradation of BRCA1 and BARD1 (lanes 3 and 4; Fig. 6C). Of note, the FSK-induced aromatase expression was partially inhibited by the MG132 treatment (compare lane 2 with 4 in Fig. 6C). Another proteasome inhibitor, ALLN, exhibited a similar effect on the aromatase level (data not shown). In light of the previously implicated role of BRCA1/BARD1 in modulating aromatase gene expression (23–25), it is likely that the stabilization of BRCA1/BARD1 by the proteasome inhibitors partially blunts the cAMP-mediated induction of aromatase expression in ovarian granulosa cells.

Fig. 6.

Accelerated Proteasome-Dependent Degradation of BRCA1/BARD1 after a cAMP Surge in Human Ovarian Granulosa Cell Line KGN A, KGN cells were treated with 25 μm FSK or 0.5 mm (Bu)₂cAMP for various time intervals as indicated on top of the panels. Cells treated with dimethylsulfoxide for 24 h served as a negative control. Whole-cell extract was analyzed by immunoblotting, using antibodies for aromatase, BRCA1, and BARD1 proteins. α-Tubulin was used as a loading control. B, Relative levels of aromatase (open circle), BRCA1 (open triangle), and BARD1 (solid square) mRNA were analyzed by quantitative real-time PCR, after treatment of KGN cells with FSK (25 μm) for various lengths of time. Glyceraldehyde-3-phosphate dehydrogenase mRNA was used for normalization. C, Levels of BRCA1 and BARD1 proteins were reduced after FSK treatment in a proteasome-dependent manner. KGN cells were treated with FSK (25 μm) for 8 h, followed by a cotreatment of FSK (25 μm) and proteasome inhibitor MG132 (5 μm) for 16 h. Whole-cell extract was prepared and analyzed by immunoblotting for the levels of aromatase, BRCA1, and BARD1 protein. D, An inverse correlation between Brca1 and aromatase expression in hCG-treated and untreated rat ovaries.

Whereas aromatase gene expression is greatly stimulated during large antral and preovulatory follicles, its level diminishes substantially after LH surge (43, 44). The inverse correlation of aromatase and BRCA1 expression in KGN cells after the cAMP activation in KGN cells prompted us to compare their expression in vivo before and after the LH surge. Immature rats were treated with pregnant mare serum gonadotropin (PMSG; 10 IU). The rats were injected 48 h after PMSG treatment with human chorionic gonadotropin (hCG; 10 IU), which mimics the effect of LH. Primary granulosa cells were harvested 6 h after the hCG treatment, and Brca1 and aromatase protein levels were analyzed by immunoblotting. As shown in Fig. 6D, decreased expression of aromatase after the hCG treatment was accompanied by an increase in the Brca1 protein level. Thus, the inverse correlation of the expression of the two proteins still stands in post-LH period.

Accumulating evidence has implicated multiple kinase pathway(s) in the cAMP-induced steroidogenic process. To interrogate the kinase pathways that mediate the cAMP-triggered degradation of BRCA1/BARD1, KGN cells were treated with forskolin in the presence of various kinase inhibitors. As shown in Fig. 7, inhibition of MAPK kinase (MEK/PD98059; lanes 5 and 6) or phosphoinoside 3-kinase (PI3K/wortmannin; lanes 7 and 8) did not prevent FSK-induced degradation of BRCA1 or BARD1. On the other hand, inhibitors of protein kinase A (PKA/H89; lanes 3 and 4) and mammalian target of rapamycin (mTOR/rapamycin; lanes 9 and 10) obliterated the FSK-induced degradation of BRCA1/BARD1. Interestingly, H89 and rapamycin also reduced the basal levels of BRCA1 and BARD1 (compare lane 1 with 3 and 9), suggesting that different kinase pathways may be involved in the regulation of the stability of BRCA1/BARD1 in cAMP-stimulated and unstimulated cells.

Fig. 7.

Treatment of KGN Cells with Pharmacological Inhibitors of Various Kinases Inhibitors for PKA (H89), MEK (PD98059), PI3K (wortmannin), and mTOR (rapamycin) were added to KGN cells treated with or without FSK. BRCA1 and BARD1 protein levels were assessed by immunoblotting 24 h after the treatment. Rapa, Rapamycin; Wort, wortmannin.

DISCUSSION

Given the paramount significance of BRCA1 in development and tumor suppression, it is evident that changes of its own expression under various physiological and pathological conditions have a significant impact on its biological activity. It has been shown that mRNA and protein abundance of BRCA1 fluctuates significantly during the cell cycle (30) and mammary gland development (19, 45, 46). In addition, BRCA1 expression is reduced in 30–40% of sporadic breast cancer (47, 48), consistent with the notion that aberrant BRCA1 expression may contribute to the development of sporadic breast cancer. More recently, BRCA1 has been implicated in the regulation of aromatase gene expression and estrogen biosynthesis in ovarian granulosa cells and adipose stromal cells (23, 24–26). A modulatory function of BRCA1 in steroidogenesis provides a potential molecular basis for the tissue-specific nature of BRCA1-associated tumors. For these reasons, we explored in the current study the underlying mechanism by which expression of BRCA1 and its partner BARD1 is regulated in ovarian granulosa cells. Our work provides evidence for the involvement of the ubiquitin-proteasome pathway in the regulation of BRCA1/BARD1 expression in granulosa cells. Importantly, the study reveals that the first 167 aa of BRCA1 are sufficient for conferring polyubiquitination and proteasome-mediated degradation. In human ovarian granulosa cells after a cAMP surge, stabilization of BRCA1/BARD1 by proteasome inhibitors is accompanied by reduced expression of aromatase, thus supporting the antagonistic relationship between BRCA1/BARD1 and aromatase expression.

The stability of BRCA1/BARD1 in the absence and presence of cAMP stimulation may be influenced by distinct signal transduction pathways. The BRCA1/BARD1 levels are low in early G₁ phase but significantly elevated in S and G₂/M phases of the cell cycle (49, 50). A recent study indicates that polyubiquitination and proteasomal degradation contribute to the cell cycle-dependent fluctuation of the steady-state level of BRCA1 protein in several cancer cell lines (30). Therefore, the basal levels of BRCA1/BARD1 in KGN cells before cAMP activation might be controlled in the same manner as the cell cycle-dependent degradation of BRCA1/BARD1 reported in other cell lines (30). However, degradation of BRCA1 in cAMP-triggered ovarian granulosa cells appears to be cell type dependent, because forskolin or (Bu)₂cAMP treatment of several other carcinoma cell lines does not lead to a significant reduction of the BRCA1 protein level (23). In addition, BRCA1 degradation in FSK-treated KGN cells is not concurrent with an increase in G₁ and/or decrease in S/G₂/M populations (data not shown), suggesting that degradation of BRCA1/BARD1 after the cAMP surge may not be cell cycle dependent. Furthermore, the differential effects of pharmacological inhibitors on the stability of BRCA1 in the FSK-treated and untreated KGN cells also implicate the involvement of distinct kinase pathways under the two circumstances.

The FSH-triggered surge in the cAMP level plays a pivotal role in ovarian folliculogenesis and steroidogenesis (27). In addition to activation of cAMP-dependent PKA, FSH is also known to stimulate other kinase pathways such as the extracellularly regulated kinase signaling pathway that requires the upstream kinase (MEK) (51) and the AKT/PI3K signaling pathway in the FSH-stimulated follicular maturation (51–57). In light of these previous reports, it is interesting to note that PKA and mTOR, but not PI3K, appear to be involved in the degradation of BRCA1/BARD1 after the FSK treatment (Fig. 7). This suggests that only a subset of the FSH-stimulated kinase pathways is required for the regulation of BRCA1/BARD1 levels in granulosa cells. One possible effect of these kinases may be to alter the subcellular localization of the BRCA1/BARD1 complex. Alternatively, phosphorylation of BRCA1/BARD1 may promote dissociation of the heterodimeric complex, which could then facilitate ubiquitination and degradation of the subunits. However, subcellular fractionation or coimmunoprecipitation of BRCA1-BARD1 after FSK treatment did not indicate any significant changes in either the relative nuclear/cytoplasmic abundance of the proteins or the protein-protein interactions (Lu, Y., and Y. Hu, unpublished data). A third possible scenario is that phosphorylation of BRCA1/BARD1 by the FSH-stimulated kinases may directly trigger ubiquitination and subsequent degradation of the proteins. Of note, BRCA1 contains multiple putative phosphorylation sites for PKA. Phosphorylation-dependent ubiquitination has been demonstrated for other important regulatory proteins (32). The exact role of various kinase pathways in BRCA1/BARD1 degradation remains to be explored in future studies.

The ubiquitin E3 ligase activity is the only biochemical function ascribed to BRCA1 so far (5). However, our study suggests that autoubiquitination is unlikely to be responsible for the proteasome-dependent degradation of BRCA1, because mutations of the key cysteine residues of the RING domain of BRCA1 that obliterate the E3 ligase activity of the protein did not stabilize the N-terminal fragment of BRCA1. Moreover, small interfering RNA-mediated depletion of endogenous BRCA1 does not affect polyubiquitination or stability of the ectopically expressed BRCA1 N-terminal fragment (Sun, J., and R. Li, unpublished data), suggesting that trans-acting autoubiquitination is unlikely to account for the proteasome-mediated degradation. Finally, BARD1 does not seem to mediate BRCA1 degradation either, as ectopic expression of BARD1 actually led to stabilization of BRCA1. We infer from these studies that the degradation-related ubiquitination event is mediated by the action of a different cellular E3 ligase. It is tempting to speculate that this putative E3 ligase may behave as a potential oncogene, because its overexpression is predicted to result in increased degradation of BRCA1/BARD1.

The exact E3 docking site(s) in the N-terminal 167-aa region of BRCA1 remains to be determined. Mutations of the highly conserved serine and threonine residues in this region neither abolish the ubiquitin ladder nor stabilize the protein fragment (Lu, Y., and Y. Hu, unpublished data). On the other hand, the simultaneous mutations of all lysine residues in this region clearly prevent the ubiquitination site and subsequent protein degradation. Of interesting note, two previous studies showed that ectopically expressed BRCA1 N-terminal fragment (1–771 aa) in HEK293T cells was polyubiquitinated, yet the conjugated species was not particularly sensitive to the proteasome-mediated degradation pathway (30, 39). This could be due to a high efficiency in the ectopic expression of the BRCA1 fragment and/or low abundance of the putative E3 ligase in HEK293T cells.

Germ line mutations of BRCA1 account for a large proportion of familial breast and ovarian cancers. The tissue specificity of BRCA1-associated tumors stands in contrast to the multiplicity of reported BRCA1 functions and its ubiquitous expression pattern. Although the activity of BRCA1 in guarding the genome integrity most likely contributes to its tumor suppressor function, loss of its DNA repair function may not be sufficient to explain why BRCA1 mutations predominantly affect estrogen-responsive tissues. The recent discovery of BRCA1 function in ovarian granulosa cells (23, 26) provides a possible molecular explanation for the longstanding conundrum. Given the well-documented function of BRCA1 in transcriptional regulation, it is conceivable that, before cAMP surge, the BRCA1/BARD1 complex may prevent the assembly and/or subsequent function of the transcription complex at the aromatase promoter. This could be achieved through the ability of BRCA1/BARD1 to ubiquitinate and degrade RNA polymerase II or other key transcription factors required for aromatase gene expression. Upon FSH-mediated cAMP elevation, degradation of the BRCA1/BARD1 complex may attenuate the inhibitory activity of the BRCA1/BARD1 complex and thus aid in the actions of transcription activators such as steroidogenic factor 1 (SF-1) to promote aromatase gene expression. In support of this model, Bulun and co-workers (24) recently showed that BRCA1 is physically associated with the endogenous aromatase promoter, and that the BRCA1 binding is diminished after the treatment of the cAMP analog (Bu)₂cAMP and phorbol diacetate (24). In the event of BRCA1 mutations or low BRCA1/BARD1 levels due to accelerated protein degradation, aberrant expression of aromatase may lead to elevated levels of either local or circulating estrogen, which may in turn increase the risk of cancer development in estrogen-responsive tissues. Therefore, a better understanding of the dynamic changes of BRCA1/BARD1 protein stability in ovarian granulosa cells may provide guidance for developing more targeted and effective therapies for BRCA1 mutation-associated cancers.

MATERIALS AND METHODS

Isolation and Analysis of Mouse Ovarian Granulosa Cell-Enriched Tissue Fractions

Ovaries were dissected out from female CD1 mice (Charles River Laboratories, Inc., Wilmington, MA) at 7 wk of age, and immersed in Dulbecco’s PBS. Large follicles were punctured with 25-gauge needles (58). Mural granulosa cell clumps were collected as they gushed out of the punctured follicles in a string-like clump of cells, and washed once in fresh PBS. COCs were collected separately and washed once in PBS. Total RNA and protein were isolated from both fractions (COC and mural granulosa cells), using TRIzol reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA) according to manufacturer’s instructions. The cDNA was synthesized with ImPromp II Reverse Transcription System (Promega Corp., Madison, WI). Quantitative real-time PCR was used to analyze the mRNA levels of Brca1, Bard1, aromatase, and a number of genes that are known to be expressed in cumulus and/or mural granulosa cells. 18s rRNA was used for normalization. The primers used in the real-time PCR will be available upon request. Immunoblotting was conducted with the following antibodies: α-BRCA1 (H-100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), α-aromatase (MCA-2077; Serotec, Oxford, UK), α-BARD1 (H-300; Santa Cruz Biotechnology), and α-tubulin (CP06; Calbiochem, La Jolla, CA).

Several criteria were used to monitor the extent of enrichment for mural and cumulus granulosa cells in these fractions. First, as shown in supplemental Fig. 3, published as supplemental data on The Endocrine Society’s Journals Online web site, the abundant expression of Fshr in the collected samples indicates an enrichment of granulosa cells, because Fshr is exclusively expressed in granulosa cells within the ovary (27, 59). Second, the preferential expression of Has2 and Lhr in the COC and mural granulosa cell samples, respectively, is consistent with previous findings (60–62). In addition, when compared with the whole ovary, the COC and mural granulosa cell samples contained very low levels of Bmp4, which is specifically expressed in thecal cells (63, 64). This suggested that the isolated fractions were not significantly contaminated with thecal cells. Finally, the fact that aromatase (Cyp19) was expressed at a high level in mural granulosa cells suggested that the fractions were enriched with granulosa cells from large antral/preovulatory follicles (i.e. before LH surge).

Human Cell Lines and Drug Treatment

The human granulosa cell line KGN is a gift from Dr. Hajime Nawata (Kyushu University, Japan) and has been previously described (29). KGN cells were grown in DMEM/F-12 Nutrient Mixture (DMEM/F12) supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin. HEK293T cells were purchased from American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin. FSK and (Bu)₂cAMP were purchased from Sigma. Proteasome inhibitor MG132 was purchased from Calbiochem. Concentrations of these reagents used in various experiments are indicated in the figure legends.

Isolation of Primary Rat Granulosa Cells

Immature (d 21 of age) Wistar female rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and kept under a 16-h light, 8-h dark regimen with food and water ad libitum. Animals were injected ip with 10 IU PMSG in 0.9% NaCl on d 22. hCG (10 IU) in 0.9% NaCl were ip injected 48 h after the PMSG injection. The animals were killed 6 h after the hCG treatment. Granulosa cells were harvested by puncturing follicles under the microscope, and the lysates were prepared immediately for immunoblotting. α-BRCA1 (H-300; Santa Cruz Biotechnology) and α-aromatase (MCA-2077; Serotec) were used.

Plasmids and Reagents

The wild-type and mutant (Δ1–95) FLAG-BARD1 expression constructs have been described previously (65). The Topo-His-HA-Ubiquitin wild-type (WT) and mutant (KO) vectors were generous gifts from Dr. Wei Gu (34). The parental mammalian expression vector pSV40-PCR was generated from pRL-SV40 (Promega) by replacing the luciferase gene from NheI to XbaI with a DNA fragment that encodes Flag, hemagglutinin (HA) tag, and a nuclear localization sequence (NLS) from simian virus 40 (SV40) large T antigen. To construct the expression vectors for various BRCA1 fragments, cDNA sequences that encode different regions of the BRCA1 protein were amplified by PCR using pcDNA3-HA-BRCA1(fl) as a template (66). The amplified fragments were inserted at the XbaI site of pSV40-PCR. To generate the untagged wild-type and mutant BRCA1 1-N (1–167 aa), the corresponding cDNA fragments were inserted between the HindIII and XbaI sites of pcDNA3 (Invitrogen). The standard PCR-mediated direct mutagenesis method was used to generate the following mutants: C61G; C64G; 11KR (K to R at position 20/32/38/45/50/55/56/65/70/109/110); 14KR (K to R at position 20/32/38/45/50/55/56/65/70/88/109/110/119/135); N4KR (K to R at position 20/32/38/45); and C6KR (K to R at position 55/56/65/70/109/110). To construct the fusion between BRCA1–1 (1–324 aa) and EGFP, a DNA fragment that encodes Flag-HA-NLS-BRCA1–1 was amplified from pSV40-PCR-BRCA1–1, and subsequently inserted between the NheI and XhoI sites of pEGFP-N2 (BD Biosciences Clontech, Genbank accession no. U57608). pCR3-Flag-BRCA1 was derived from pCR3-BRCA1, a gift from Barbara L Weber (67), by inserting an oligonucleotide that encodes the Flag tag at the beginning of the coding region. pCR3-Flag-ΔN-BRCA1 that lacks the first 906 nucleotides of the BRCA1 coding sequence was made by digesting pCR3-Flag-BRCA1 with HindIII and EcoRI and replacing the fragment with an oligonucleotide that encodes the Flag tag. All subcloned DNA sequences were verified by DNA sequencing.

Transient Transfection

Transient transfection in KGN cells was carried out with the FuGene 6 transfection reagent (Roche Diagnostics Corp., Indianapolis, IN) according to manufacturer’s instruction. Briefly, KGN cells were plated in growth medium free of antibiotics 24 h before transfection. Cells were transfected in the presence of serum and collected for the expression analysis 24 h post transfection. For those experiments that involved the treatment of FSK or MG132, transfected cells were split 1:2 4–6 h after transfection. After an overnight incubation, the transfected cells were treated with FSK or MG132 for 12 h. Transient transfection in HEK293T cells was performed with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instruction.

RNA Isolation and Quantitative RT-PCR Assay for Tissue Culture Cells

Total RNA was isolated with Trizol reagent (Invitrogen) according to manufacturer’s instruction. Total RNA (1 μg) was used for cDNA synthesis with the ImPromp II kit (Promega). The mRNA levels of various genes were determined by quantitative real-time PCR analysis, using the SYBR Green technology and an ABI7300 thermo-cycler (Applied Biosystems, Foster City, CA). The primers used in the real-time PCR analysis were designed with the Primer Express software (Applied Biosystems) and will be provided upon request. The relative expression levels of different genes were normalized against those of β-actin or glyceraldehyde-3-phosphate dehydrogenase.

Human Cell Extract Preparation and Immunoblotting

Whole-cell extracts were made with Lysis Buffer (50 mm Tris, pH 6.8; 2% sodium dodecyl sulfate; 10% glycerol), and protein concentration was determined with the BCA assay (Pierce Chemical Co., Rockford, IL). An equal amount of protein extract was resolved by SDS-PAGE. Western Blot analysis was carried out using the following antibodies: α-BRCA1 Ab1 (OP92; Calbiochem), α-BRCA1 Ab5 (OP116; Calbiochem), α-aromatase (MCA2077; Serotec), α-tubulin (CD06; Calbiochem), α-lamin A&C (sc-7292; Santa Cruz Biotechnology), α-Flag (F3165; Sigma), and α-HA (A190–108A; Bethyl Laboratories, Montgomery, TX). The polyclonal α-BARD1 antibody 59M was raised against a glutathione-S-transferase-fusion protein containing residues 141–388 of human BARD1. Peroxidase-conjugated α-mouse IgG and α-rabbit IgG antibody (31430, 31460; Pierce) were used as the secondary antibodies for Western blotting. Blots were visualized with the enhanced chemiluminescence method (Pierce).

Detection of Ubiquitination

Approximately 2 × 10⁶ of HEK293T cells were transfected with 4 μg of pcDNA3-BRCA1–1-N (or 1-N-14KR) and 4 μg of Topo-His-HA-Ubiquitin wild type or KO. MG132 was added to the transfected cells at a final concentration of 5 μm 36 h after transfection, and the cells were incubated for another 12 h before harvest. Cells were then lysed in a phosphate/guanidine buffer with mild sonication (6 m guanidine-HCl; 10 mm Tris-HCl, pH 8.0; 50 mm Na₂HPO₄/NaH₂PO₄, pH 6.4; 100 mm NaCl; 10 mm imidazole; with freshly added 10 mm β-mercaptoethanol and 10 mmN-ethylmaleimide). The ubiquitinated proteins were precipitated with Ni-NTA agarose (QIAGEN, Chatsworth, CA), followed by four washes with 8 m urea buffer (8 m urea; 50 mm Na₂HPO₄/NaH₂PO₄, pH 8.0; 100 mm NaCl). The precipitated proteins were eluted with NTA elution buffer (150 mm Tris-HCl, pH 6.8; 200 mm imidazole; 5% sodium dodecyl sulfate; 30% glycerol; 0.72 m β-mercaptoethanol), resolved by 17% SDS-PAGE, and analyzed by immunoblotting with the α-BRCA1 Ab1.

Acknowledgments

We thank Drs. Hajime Nawata, Wei Gu, Barbara Weber, and Beric Henderson for various plasmids used in the study.

This work was supported by a predoctoral fellowship (W81XWH-06-1-0302) from Department of Defense Breast Cancer Research Program (to J.S.), a grant from Henrietta Milstein Foundation (to R.B.), and National Institutes of Health Grants CA118578 (to Y.H.) and CA93506 (to R.L.).

Disclosure Statement: The authors have nothing to disclose.

Abbreviations

 
• aa

  Amino acids;

 
• (Bu)₂cAMP

  N,O′-dibutyryl-cAMP;

 
• CG

  chorionic gonadotropin;

 
• COC

  cumulus-oocyte complex;

 
• EGFP

  enhanced green fluorescent protein;

 
• ER

  estrogen receptor;

 
• FSK

  forskolin;

 
• HA

  hemagglutinin;

 
• HEK

  human embryonic kidney;

 
• His-Ub

  histidine-tagged ubiquitin;

 
• KO

  knockout;

 
• MEK

  MAPK kinase;

 
• mTOR

  mammalian target of rapamycin;

 
• NLS

  nuclear localization sequence;

 
• PI3K

  phosphoinoside 3-kinase;

 
• PKA

  protein kinase A;

 
• PMSG

  pregnant mare serum gonadotropin;

 
• SV40

  simian virus 40.