Inhibition of histone deacetylases targets the transcription regulator Id2 to attenuate cystic epithelial cell proliferation

Abstract

The pan-histone deacetylase (HDAC) inhibitor, trichostatin A, was found to reduce cyst progression and slow the decline of kidney function in Pkd2 knockout mice, model of autosomal dominant polycystic kidney disease (ADPKD). Here we determine whether HDAC inhibition acts by regulating cell proliferation to prevent cyst formation, or by other mechanisms. The loss of Pkd1 caused an upregulation of the inhibitor of differentiation 2 (Id2), a transcription regulator, triggering an Id2-mediated downregulation of p21 in mutant mouse embryonic kidney cells in vitro. Using mouse embryonic kidney cells, mutant for Pkd1, we found that trichostatin A decreased Id2, which resulted in upregulation of p21. Further, phosphorylated retinoblastoma (Rb), usually regulated by Cdk2/Cdk4 activity, was also reduced in these cells. Since these latter enzymes are under the control of p21, these studies suggest that the proliferation of cyst epithelial cells that is reduced by trichostatin A might result from p21 upregulation, or alternatively through the Rb-E2F pathway. Additional studies showed that Id2 directly bound to Rb, releasing the transcription activator E2F from transcriptionally inactive Rb-E2F complexes. HDAC inhibition was able to reverse this process by downregulation of Id2. Furthermore, treatment of pregnant Pkd1 mice with trichostatin A prevented cyst formation in the developing embryonic kidneys, showing that this inhibition is effective in vivo during early cyst formation. Thus, HDAC inhibition targets Id2-mediated pathways to downregulate cystic epithelial cell proliferation and hence cystogenesis.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common hereditary disorders in humans, affecting 1 in 500–1000 in the United States.¹ The hallmark of the disease is the development of multiple bilateral renal cysts that replace the normal renal parenchyma, resulting in end-stage renal disease in ~50% of individuals with ADPKD. Cyst formation is thought to start early in kidney development and continue throughout the life of the affected individual. Most cases of ADPKD are caused by mutations in one of two genes: PKD1, accounting for 85–95% of the cases, and PKD2, accounting for most of the remainder.² The gene product of PKD2, polycystin-2 (PC2), either alone or in complex with the gene product of PKD1, polycystin-1 (PC1), appears to function as a calcium-permeable cation channel.^3–6 The unexpected association of the primary cilium with several inherited cystic kidney diseases and localization of cysto-proteins including PC1 and PC2 to cilia has led to the ‘primary cilia’ hypothesis. Simply stated, the hypothesis is that structural or functional abnormalities in the primary apical cilia of tubular epithelia have a role in renal cyst development and may represent a unifying mechanism of cyst formation.

In addition to well-characterized genetic abnormalities that lead to cyst initiation and progression, it is now recognized that epigenetic regulators may also have a significant role in cystogenesis. It has been shown recently that histone deacetylases (HDACs) may be promising targets for ADPKD treatment.^7,8 It has been found that inhibiting HDACs either by a class I HDAC inhibitor, valproic acid, or by a pan-HDAC inhibitor, trichostatin A (TSA), reduces the cyst progression and slows the decline of kidney function in Pkd1 and Pkd2 knockout mice. However, the underlying mechanisms remain to be clarified.

HDACs regulate cellular functions either through deacetylation of histones or nonhistone transcription factors.^9–12 In addition to the more global effects of HDACs on gene expression through deacetylation of histones, HDACs may also affect unique pathways through specific HDAC-targeted proteins to regulate specific cellular processes. In this study, we report that HDAC inhibition (HDACi) targets inhibitor of differentiation 2 (Id2), thus affecting Id2-mediated p21 and Rb–E2F pathways to regulate cystic epithelial cell proliferation. We have found that loss of Pkd1 leads to upregulation of Id2, and that HDACi decreases Id2 levels, causing upregulation of p21 in Pkd1 mutant cells. HDACi also decreased the levels of phosphorylated retinoblastoma (Rb), which was regulated by p21-mediated Cdk2/Cdk4 activity, suggesting that the upregulated p21 might work through the Rb–E2F pathway to inhibit cystic epithelial cell proliferation. Id2 was also found to function by directly binding and sequestering Rb, thus releasing E2F from Rb–E2F complexes, and that HDACi was able to reverse this process. Treatment with TSA delayed cell cycle reentry in Pkd1 mutant mouse embryonic kidney (MEK) cells. Furthermore, treatment of pregnant mice with TSA prevented cyst formation in Pkd1 mutant embryonic kidneys, demonstrating that HDACi is effective during early cyst development. These findings suggest that HDACi is a potential therapeutic approach for treating ADPKD.

RESULTS

HDACi reduces S-phase entry of Pkd1 mutant MEK cells

Although it has been recognized recently that HDACs may be useful targets for ADPKD treatment,^7,8 the underlying mechanisms are unclear. Previous studies using inhibitors of HDACs in cancer cells indicated that these compounds reduce cell proliferation. Therefore, we examined whether addition of the HDAC inhibitor TSA to MEK cells produced any effect on cell proliferation. We found that TSA-treated (100 ng/ml) Pkd1 mutant (Pkd1^null/null) MEK cells proliferated ~60% slower than nontreated Pkd1^null/null MEK cells. We also found that inhibition of HDACs with TSA was cell cycle dependent (Table 1). By fluorescence-activated cell sorting analysis, we could show that about 62%, 29%, and 9% of the TSA-treated Pkd1^null/null MEK cells were in G1, S, and G2 phases, respectively. By contrast, about 43%, 47%, and 9% of the control (untreated) Pkd1^null/null MEK cells were in G1, S, and G2 phases, respectively. The cell cycle profile of the TSA-treated Pkd1^null/null MEK cells was similar to that of the wild-type MEK cells in that about 67%, 27%, and 6% of the wild-type cells were in G1, S, and G2 phases, respectively. The differences of the cells in G0/G1 and in S-phase between the Pkd1 mutant MEK cells and the cells including Pkd1 mutant MEK cells treated with TSA and Pkd1 wild-type MEK cells treated with or without TSA were statistically significant (P<0.05), which suggested that TSA treatment might restore the cell cycle profile of Pkd1^null/null MEK cells by decreasing S-phase entry.

Table 1.

TSA treatment decreased S-phase entry of Pkd1 mutant MEK cells

Pkd1	G0/G1 phase (%)	S phase (%)	G2 phase (%)
WT	66.7 ± 8.4	27.3 ± 5.5	6.0 ± 3.1
WT-TSA	66.7 ± 5.9	28.3 ± 7.3	4.7 ± 1.3
Null	42.7 ± 2.9	47.3 ± 5.3	9.0 ± 2.9
Null-TSA	61.7 ± 3.0	29.0 ± 1.7	9.3 ± 1.3

Abbreviations: FACS, fluorescence-activated cell sorting; MEK, mouse embryonic kidney; TSA, trichostatin A; WT, wild type.

Data present mean ± s.e. calculated from three independent FACS analyses.

The differences of the cells in G0/G1 and in S-phase between the Pkd1 mutant MEK cells (null cells) and the other cells, which including Pkd1 WT MEK cells treated with or without TSA and Pkd1 mutant MEK cells treated with TSA, were statistically significant (P< 0.05). In particular, the S-phase entry of Pkd1 mutant MEK cells (null cells) were significantly increased compared with other cells, whereas TSA treatment significantly decreased S-phase entry of Pkd1 mutant MEK cells (P<0.01).

HDACi decreases expression of Id2 and increases expression of p21 in Pkd1 mutant MEK cells

Expression of the key cell cycle regulator p21 has been shown to be decreased in Pkd1 mutant MEK cells and Pkd1 mutant mouse kidneys.^13,14 We found that treatment with TSA increased the expression of p21 in Pkd1^null/null MEK cells to the levels seen in Pkd1 wild-type MEK cells (treated or untreated with TSA; P<0.01; Figure 1a). To determine the potential mechanisms for this increased expression of p21 in response to TSA treatment, we examined the expression of Id2 and HDAC1, two key regulators of p21 transcription, in other cells.^15,16 We found that Id2 expression was upregulated in Pkd1^null/null MEK cells and that TSA treatment decreased the expression of Id2 in these cells to the levels seen in Pkd1 wild-type MEK cells (P<0.001; Figure 1a). In contrast, the expression levels of HDAC1 were not affected by TSA treatment (Figure 1a). In addition, the levels of Id2 in Pkd1^null/null MEK cells over a number of hours after treatment with TSA gradually decreased, over the same time the levels of p21 increased (Figure 1b). These results suggested that TSA might regulate the expression of p21 through Id2.

Figure 1. Trichostatin A (TSA) decreased the expression of inhibitor of differentiation 2 (Id2) but increased the expression of p21 in Pkd1^null/null mouse embryonic kidney (MEK) cells.

(a) Western blot analysis of the expression of Id2, p21, and histone deacetylase (HDAC) 1 from whole-cell lysates of Pkd1 wild-type (WT) and Pkd1^null/null MEK cells treated with or without TSA. The expression of Id2 was decreased in Pkd1^null/null MEK cells treated with TSA, whereas the expression of p21 was increased in Pkd1^null/null MEK cells after TSA treatment. The expression of Id2 and p21 with or without TSA treatment in MEK cells was quantified from three independent immunoblots by densitometric analysis and was presented as a ratio of the band density of Id2 or p21 to actin. P value was marked in the graph (bottom panels). The expression of HDAC1 was not affected by TSA treatment. (b) Western blot analysis of the expression of Id2 and p21 from whole-cell lysates of Pkd1^null/null MEK cells treated with TSA at 0, 2, 4, 8, 12, and 24 h. Decreased expression of Id2 is accompanied by increased expression of p21 in Pkd1^null/null MEK cells.

Id2, but not HDAC1, regulates the expression of p21 in Pkd1^null/null MEK cells

To determine whether the expression of p21 is regulated by Id2 in Pkd1 mutant MEK cells, Id2 was knocked down with small interfering RNA (siRNA) and p21 expression was examined. We found that inhibition of Id2 by siRNA led to increased p21 expression (P<0.01; Figure 2a).

Figure 2. Inhibitor of differentiation 2 (Id2), but not histone deacetylase (HDAC) 1, regulates the expression of p21 in Pkd1^null/null mouse embryonic kidney (MEK) cells.

(a) Transfection of small interfering RNA (siRNA) against Id2 into MEK cells inhibited Id2 expression and also resulted in increased expression of p21 in Pkd1^null/null MEK cells but not in Pkd1 wild-type (WT) MEK cells. The expression of p21 with or without knockdown Id2 with siRNA in MEK cells was quantified from three independent immunoblots by densitometric analysis and was presented as a ratio of the band density of p21 to actin. P value was marked in the graph (right panels). (b) Chromatin immunoprecipitation (ChIP) assay demonstrated that HDAC1 bound to the promoter region of p21 in MEK cells treated or untreated with trichostatin A (TSA). Pkd1 WT and Pkd1^null/null MEK cells were treated with or without TSA, and an anti-HDAC1 antibody was used for ChIP analysis. PCR was performed using primers in the p21 promoter region as explained in Materials and Methods. Input is the DNA template before ChIP. Binding of HDAC1 appeared to be reduced in samples of Pkd1 WT MEK cells treated with TSA compared with samples of Pkd1^null/null MEK cells. Anti-IgG was used as a negative control. (c) Transfection of siRNA against HDAC1 into MEK cells inhibited HDAC1 expression but did not affect the expression of p21 in Pkd1 WT and Pkd1^null/null MEK cells. IgG, immunoglobulin G.

Although HDAC1 expression did not appear to be affected by TSA treatment, TSA might still affect HDAC1 function or activity. To test this possibility, we examined binding of HDAC1 to the p21 promoter by chromatin immunoprecipitation. As shown in Figure 2b, we found that HDAC1 bound to the p21 promoter in both Pkd1 wild-type and Pkd1^null/null MEK cells. Although we observed that binding of HDAC1 to the p21 promoter was decreased in Pkd1 wild-type MEK cells treated with TSA, the expression levels of p21 were not changed (Figure 1a). In addition, inhibition of HDAC1 with siRNA did not lead to activation of p21 expression (Figure 2c). These results suggest that HDACi through targeting Id2, but not HDAC1, regulates the expression of p21 in wild-type and Pkd1^null/null MEK cells.

TSA affects the Rb–E2F1 pathway

Fluorescence-activated cell sorting analysis suggested that S-phase entry is significantly increased in Pkd1 mutant MEK cells and that TSA treatment decreases this S-phase entry (Table 1), thus slowing cell proliferation. Id2 has been found to stimulate cell proliferation by two mechanisms: (1) by direct binding to Rb and (2) by downregulation of p21, resulting in increased Rb phosphorylation. In both cases, Rb is released from the Rb–E2F complex, allowing E2F to activate target cell cycle genes.¹⁵ We found that Rb, phospho-Rb, and E2F1 were upregulated in Pkd1^null/null MEK cells compared with wild-type cells (Figure 3a). We also found that TSA significantly decreased expression of phospho-Rb (P<0.01; Figure 3a, bottom panel), but only slightly decreased expression of Rb and E2F1 in Pkd1^null/null MEK cells (Figure 3a). We next examined the interactions between Id2 and Rb and between Rb and E2F1 by co-immunoprecipitation analysis. We found that anti-Id2 and anti-E2F antibodies could pull down Rb from cell lysates with or without TSA, and anti-Rb antibody could also pull down E2F1 (Figure 3b). As expected, anti-phospho-Rb antibody could not pull down E2F1 nor could anti-Id2 antibody pull down phospho-Rb (Figure 3b), which suggested that phospho-Rb could not bind with Id2 and E2F1 in kidney epithelial cells.

Figure 3. Trichostatin A (TSA) targets the Rb–E2F pathway.

(a) Retinoblastoma (Rb), phospho-Rb, and E2F1 are upregulated in Pkd1^null/null mouse embryonic kidney (MEK) cells. The expression of Rb, phospho-Rb, and E2F1 in Pkd1 wild-type (WT) and Pkd1^null/null MEK cells treated with or without TSA was analyzed by western blotting. The expression of Rb, phospho-Rb, and E2F1 were upregulated in Pkd1^null/null MEK cells compared with Pkd1 WT MEK cells untreated with TSA. After TSA treatment, phospho-Rb was decreased to the same level seen in Pkd1 WT MEK cells either treated with or without TSA. The expression of phospho-Rb with or without TSA treatment in MEK cells was quantified from three independent immunoblots by densitometric analysis and was presented as a ratio of the band density of phospho-Rb to actin. P value was marked in the graph (bottom panel). (b) Co-immunoprecipitation assay indicated that Rb, but not phospho-Rb, co-immunoprecipitated with inhibitor of differentiation 2 (Id2) and E2F1 in MEK cells treated or untreated with TSA. Anti-Id2 antibody pull down Id2 always produced a doublet, whereas only the lower band represented Id2 as reported previously.¹³ (c) Co-immunoprecipitation assay indicated that only Rb, but not phospho-Rb, co-immunoprecipitated with histone deacetylase (HDAC) 1 in MEK cells treated or untreated with TSA. IP, immunoprecipitation; IB, immunoblotting.

It has been reported that HDAC1 can form a trimeric complex with Rb-E2F to repress gene expression.^17,18 To test for the presence of this complex, with or without TSA treatment in Pkd1^null/null MEK cells, we carried out co-immunoprecipitation experiments. We found that HDAC1 could be pulled down with anti-Rb antibody irrespective of whether cells were treated with TSA (Figure 3c). However, phospho-Rb could not be pulled down with anti-HDAC1 antibody.

PC1 affects the expression of Id2 in kidney epithelial cells

To exclude the possibility that the upregulation of Id2 in Pkd1^null/null MEK cells was being selected for or caused by culturing under conditions that favor upregulation of Id2, we performed the following two experiments. First, we knocked down Pkd1 with either oligonucleotide-mediated siRNA (Figure 4a) or lentiviral vector-mediated siRNA (Figure 4b) in mouse inner medullary collecting duct cells and found that these resulted in upregulation of Id2. We also found that c-Myc was upregulated (Figure 4a). Second, when Pkd1 was transfected back into Pkd1^null/null MEK cells, it reduced the expression of Id2 and c-Myc (Figure 4c). These experiments suggested that the upregulation of Id2 in these cells was due to the loss of Pkd1. As our anti-PC1 antibody could not directly detect PC1 in western blotting analyses, we carried out immunoprecipitation experiments with this anti-PC1 antibody, followed by western blotting to detect the presence of PC1 (Figure 4a and c). We also evaluated the efficiency of Pkd1 knockdown by RT-PCR (Figure 4a and b). It has been reported that c-Myc could regulate the expression of Id2.^19,20 We further found that knockdown of c-Myc with siRNA decreased the expression of Id2 in Pkd1 mutant MEK cells (Figure 4d), which suggested that c-Myc was at least one of the factors, together with HDACs, involved in regulating the expression of Id2 in Pkd1 mutant kidney epithelial cells.

Figure 4. Polycystin-1 (PC1) affects the expression of inhibitor of differentiation 2 (Id2) in kidney epithelial cells.

(a) Transfection of small interfering RNA (siRNA) against PC1 into mouse inner medullary collecting duct (IMCD) cells inhibited PC1 expression and resulted in upregulation of Id2 and c-Myc. The expression of PC1 was detected by immunoprecipitation and then immunoblotting analysis with an anti-PC1 antibody and also examined by RT-PCR. (b) PC1 expression was also knocked down by the constitutive expression of specific anti-Pkd1 siRNA mediated by a puromycin-selectable lentiviral vector, VIRHD/P/siPKD1³²⁹⁷, which strongly increased the expression of Id2 compared with the control VIRHD/P/siLuc lentivector-transduced cells. Mouse IMCD3 cells were transduced with VIRHD/P/siPKD1³²⁹⁷ or the control VIRHD/P/siLuc lentivector, in which the Pkd1 siRNA was replaced by the anti-luciferase siRNA.²⁴ PC1 expression in VIRHD/P/siPKD1³²⁹⁷ was reduced by more than 90% when compared with the transduced control VIRHD/P/siLuc lentivector, as assayed by reverse transcription-PCR (bottom panels). (c) Transient transfection of Pkd1 into Pkd1^null/null mouse embryonic kidney (MEK) cells reduced the expression of Id2 and c-Myc. The expression of PC1 was detected by immunoprecipitation and then by immunoblotting analysis with an anti-PC1 antibody. (d) Transfection of siRNA against c-Myc into Pkd1 mutant MEK cells inhibited c-Myc expression and resulted in downregulation of Id2.

TSA prevents cyst formation in Pkd1 mutant embryonic kidneys

It has been reported that TSA treatment reduces the progression of cyst formation in Pkd2 knockout mice.⁷ However, whether TSA can suppress cyst formation in Pkd1 knockout mice is unknown. To test this possibility, we injected TSA into pregnant Pkd1^m1Bei+/– female mice from 8.5 to 14.5 days postcoitus (d.p.c.). The embryonic kidneys were then analyzed at 15.5 d.p.c. In all Pkd1^m1Bei–/– embryos from TSA-injected mothers, kidney cyst formation was significantly prevented or reduced compared with that from dimethyl sulfoxide-injected mothers (P<0.05; Figure 5a–e). Furthermore, we found that the expression of Id2 from the TSA-treated Pkd1^m1Bei–/– embryonic kidneys was decreased compared with that from the dimethyl sulfoxide-treated Pkd1^m1Bei–/– embryonic kidneys (control) by western blot analysis (Figure 5f) and by immunohistochemistry analysis (Figure 5g, top panels). In contrast, the expression of Id2 was increased in the cyst-lining epithelia without TSA treatment by immunostaining with anti-Id2 antibody (Figure 5g, bottom panels). We further found that double knockout Id2 and Pkd1 also prevented cyst formation in E15.5 embryonic kidneys (Figure 5h), which strongly supported the critical role of Id2 in this process.

Figure 5. Treatment with trichostatin A (TSA) prevents cyst formation in Pkd1^m1Bei–/– embryonic kidneys.

(a–d) Representative histological sections of E15.5 Pkd1^+/+ and Pkd1^m1Bei–/– embryonic kidneys from pregnant mothers injected with TSA or control vehicle (dimethyl sulfoxide, DMSO) from 8.5 to 14.5 d.p.c. (a) Pkd1^+/+ from TSA-treated mother; (b) Pkd1^+/+ from DMSO-treated mother; (c) Pkd1^m1Bei–/– from TSA-treated mother; and (d) Pkd1^m1Bei–/– from DMSO-treated mother. (e) Aggregate data from vehicle-treated (n = 5) and TSA-treated (n = 6) mouse embryonic kidneys showing significant differences in cystic index (P<0.05). Quantification of the percentage of cystic areas over total kidney section areas of Pkd1 wild-type (WT) and Pkd1^m1Bei–/– treated with or without TSA. The middle section of each kidney was quantified for all mice under each condition. Shown are mean and s.e.m. of all sections quantified for each condition. Treatment with TSA significantly prevents cyst formation in Pkd1^m1Bei–/– embryonic kidneys compared with the control to the left (P<0.05). (f) Western blot analysis of the expression of inhibitor of differentiation 2 (Id2) from Pkd1^+/+ (WT) or Pkd1^m1Bei–/– embryonic kidneys treated with or without TSA. The expression levels of Id2 were decreased in Pkd1^m1Bei–/– embryonic kidneys treated with TSA. (g) Immunohistochemistry (top panel) and immunostaining (bottom panel) analyses of the expression of Id2 from Pkd1^+/+ or Pkd1^m1Bei–/– embryonic kidneys treated with DMSO or TSA. (h) Knockout of Id2 rescues renal cystic phenotype in Pkd1^null/null mutants. Id2-Pkd1 double knockout (KO) and Id2^+/+:Pkd1^null/null embryonic kidneys are displayed.

DISCUSSION

Our study provides new insights into the mechanisms of ADPKD by uncovering a network of HDAC signaling through targeting Id2 and Id2-mediated downstream p21 and Rb–E2F pathways to modulate the proliferation of cystic epithelial cells. We found that Id2 was upregulated in Pkd1 mutant and Pkd1 knockdown kidney epithelial cells. Our fluorescence-activated cell sorting analysis suggested that S-phase entry is significantly increased in Pkd1 mutant MEK cells (Table 1). We propose that upregulated Id2 may increase S-phase entry in Pkd1 mutant MEK cells (1) through binding with Rb to control Rb-E2F-mediated S-phase entry; (2) through binding with basic helix–loop–helix transcriptional factors to inhibit transcription of the p21 gene; and (3) through inhibition of p21 to increase Cdk2/Cdk4-mediated phosphorylation of Rb, which will also release E2F from the Rb–E2F complex to increase S-phase entry. We found that HDACi decreased the expression of Id2 in Pkd1 mutant MEK cells (Figure 1a). As such, HDACi might (1) free Rb from Id2–Rb complexes, which would increase Rb–E2F1 complex to decrease the E2F1-mediated S-phase entry; (2) release the inhibition of Id2 on p21 gene transcription, which would increase the expression of p21 (Figures 1a and 2a) and decrease p21-mediated S-phase entry; and (3) through increased p21 to decrease p21-Cdks-mediated phosphorylation of Rb (Figure 3a), which would also increase the formation of Rb–E2F1 complex to decrease the E2F1-mediated S-phase entry (summarized in Figure 6). This study not only furthers our previous finding that PC2 interacts with Id2 in a PC1-dependent manner, sequestering Id2 in the cytoplasm to regulate kidney epithelial cell proliferation,¹³ but also provides the mechanism of HDACi in preventing cyst formation in Pkd1 knockout mouse models.^7,8

Figure 6. A schematic diagram depicting a pathway that connects polycystins (PCs) and histone deacetylase (HDAC) inhibitor to inhibitor of differentiation 2 (Id2) and Id2-mediated p21 and Rb–E2F pathways in regulating cystic epithelial cell proliferation.

Pkd1 knockout or mutation results in the upregulation of Id2. We propose that upregulated Id2 may increase S-phase entry in Pkd1 mutant mouse embryonic kidney cells (1) through binding with retinoblastoma (Rb) to control Rb-E2F-mediated S-phase entry; (2) through binding with basic helix–loop–helix (bHLH) transcriptional factors to inhibit transcription of the p21 gene; and/or (3) through inhibition of p21 to increase Cdk2/Cdk4-mediated phosphorylation of Rb, which will also release E2F from Rb–E2F complex to increase S-phase entry. HDAC inhibition decreased the expression of Id2 and would further (1) free Rb from Id2–Rb complexes, which would increase Rb–E2F1 complex to decrease the E2F1-mediated S-phase entry; (2) release the inhibition of Id2 on p21 gene transcription, which would increase the expression of p21 and decrease p21-mediated S-phase entry; and (3) through increased p21 to decrease p21-Cdks-mediated phosphorylation of Rb, which would also increase the formation of Rb–E2F1 complex to repress E2F1-mediated S-phase entry. This diagram also incorporates our previous finding that the PC2–Id2 interaction regulates the shuttling of Id2 between nuclear and cytosolic compartments and that this process is regulated by PC1. TSA, trichostatin A; VPA, valproic acid.

HDAC inhibitors have been found to repress cyst formation in ADPKD mouse models.^7,8 However, whether HDACi functions by regulating cell proliferation to prevent cyst formation, or functions by some other mechanisms, is unknown. To identify the specific targets of HDACi in kidney epithelial cell cycle regulation, we examined the expression of p21 and its regulators, Id2 and HDAC1. HDAC1, through binding to the p21 promoter, has been found to repress p21 expression in cancer cells.¹⁶ Id2, through binding to basic HLH proteins, has also been found to regulate p21 expression.¹⁵ We found that the expression of p21 in response to HDACi was correlated with expression of Id2, but not HDAC1, in TSA-treated Pkd1 mutant MEK cells (Figure 1b). TSA did not affect Id2 or p21 in Pkd1 wild-type MEK cells. We then confirmed that Id2 was responsible for regulation of p21 expression by Id2 siRNA knockdown (Figure 2a). In addition, we excluded the possibility that HDACi functions through HDAC1 to regulate p21 gene expression in Pkd1^null/null MEK cells by examining the effect of TSA on binding of HDAC1 to the p21 promoter (Figure 2b) and by HDAC1 siRNA knockdown (Figure 2c). Thus, our results indicate that Id2 is not only the target of HDACi but also the key regulator of p21 expression in Pkd1 mutant kidney epithelial cells, which suggests that HDACi may be through targeting Id2–p21 pathway to regulate the cystic epithelial cell cycle.

Id2 has also been reported to regulate cell proliferation through the Rb–E2F pathway.¹⁵ Id2 can regulate the formation or function of the Rb–E2F complex either by directly binding with Rb or through p21-Cdks-mediated phosphorylation of Rb, thus releasing E2F from the Rb–E2F complex and allowing S-phase-specific genes to be transcribed.¹⁵ We found that phospho-Rb was upregulated in Pkd1^null/null MEK cells and that TSA decreased the upregulated phospho-Rb to levels seen in wild-type MEK cells (Figure 3a). In addition, the upregulated Id2 and Rb appear to be bound to each other (Figure 3b), which would also release E2F from the inhibition of Rb. The decreased expression of p21 in Pkd1 mutant MEK cells would be expected to decrease its inhibition of Cdks, and might be responsible for increasing phospho-Rb, releasing E2F from the Rb–E2F complex (Figure 6).

Many of the genes that are required for DNA synthesis contain E2F sites in their promoters. Transcription from promoters containing E2F sites can be actively repressed by a trimeric complex containing E2F, Rb, and HDAC1 or HDAC2.^17,18,21 Before the G1–S transition, phosphorylation of Rb leads to dissociation of the E2F–Rb–HDAC repressor complex. E2F is then free to activate transcription by contacting basal factors or histone acetyltransferases, such as CREB-binding protein, which can alter chromatin structure. We found that HDAC1 interacts with Rb but not phospho-Rb in Pkd1 wild-type and Pkd1^null/null MEK cells, either treated with TSA or untreated (Figure 3c). These results suggested that although HDAC1 may not be involved in regulating the expression of p21 in these cells, it may still be involved in regulating other genes through the Rb–E2F pathway (Figure 6).

In the current working model, we also provided evidence to support a causative role for PC1 in regulating the expression of Id2 in kidney epithelial cells through Pkd1 knockdown and rescue experiments (Figure 4). In our previous study, we found that Id2 expression was upregulated in the kidney of ADPKD patients, but we did not find a significant difference in Id2 expression between Pkd1 wild-type and mutant MEK cells.¹³ However, in this study, we found that Id2 was upregulated in Pkd1 mutant MEK cells. The different cell growth conditions might be the cause of this difference. In particular, all MEK cells used in this study were synchronized by serum starvation and then refed with serum for further analysis.

Our results and the published evidence suggested that c-Myc together with HDACs was involved in regulating the expression of Id2 in kidney epithelial cells. It has been found that in ADPKD, renal c-Myc expression is consistently elevated up to 15-fold.²² c-Myc has also been shown to regulate the expression of Id2,^19,20 and together with HDACs has been shown to regulate the transcription of a number of genes.²³ We found that when Pkd1 was knocked down with its siRNA, the expression of c-Myc was upregulated, and when Pkd1 was transiently transfected back into Pkd1 mutant MEK cells, the expression of c-Myc was down-regulated (Figure 4). We further found that knockdown of c-Myc with siRNA decreased the expression of Id2 in Pkd1 mutant MEK cells (Figure 4d), which suggested that c-Myc was at least one of the factors, together with HDACs, involved in regulating the expression of Id2 in Pkd1 mutant kidney epithelial cells.

Valproic acid, the class I HDAC inhibitor, was found to repress cyst formation in Pkd1 conditional knockout mice.⁸ These results suggest that class I HDACs are involved in regulating cystogenesis. Although the expression of class I HDACs was not changed, valproic acid might affect the activity of class I HDAC, such as HDAC1, to regulate the pathway(s) downstream of it, such as HDAC1-Rb-E2F in some way as we presented in the current model (Figure 6). However, this possibility needs to be further investigated. In this study, we found that the pan-HDAC inhibitor, TSA, prevented cyst formation in vivo in Pkd1 knockout embryonic kidneys (Figure 5), very possibly due to targeting Id2 and Id2-mediated p21 and Rb–E2F pathways. Id2 and Pkd1 double knockout prevented cyst formation and further supported the central role of Id2 in this process (Figure 5h). As such, our findings provide mechanism of HDACi in preventing cyst formation in Pkd1 knockout mouse model and further support the hypothesis that HDAC inhibitors are potential therapeutic agents for the treatment of ADPKD.

MATERIALS AND METHODS

Cell culture and cell cycle analysis

Pkd1 wild-type and Pkd1^null/null MEK cells were maintained as described previously.¹³ For cell cycle analysis, MEK cells were synchronized by serum starvation for 24 h, followed by addition of 2% serum with or without TSA for 48 h, stained with propidium iodide, and analyzed by fluorescence-activated cell sorting. For all other analysis, MEK cells were also synchronized by serum starvation for 24 h, followed by refeeding with 2% serum.

Immunoprecipitation and western blotting

Immunoprecipitation and western blotting were performed on whole-cell lysates as described by the manufacturer (Upstate Biotechnology, Lake Placid, NY). The antibodies used for western blotting included anti-Id2 (c-20), p21 (F-5), E2F1 (c-20), Rb (c-15), and PC1 (H260) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); anti-HDAC1 antibodies (Upstate-Millipore, Lake Placid, NY); anti-actin, anti-tubulin, and anti-acetyl-tubulin antibodies (Sigma, St Louis, MO); and anti-phospho-Rb (Cell Signaling Technology, Beverly, MA). All primary antibodies were used at 1:50 for immunoprecipitation and 1:500 for western blotting. Donkey anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase and Donkey anti-mouse IgG-horseradish peroxidase (1:8000 dilution, Santa Cruz) were used as secondary antibodies for western blotting.

Chromtin immunoprecipitation

Chromatin was prepared from Pkd1 wild-type and Pkd1^null/null MEK cells treated with or without TSA for 24 h according to the manufacturer's instructions (Upsate), with a crosslinking time of 15 min at 25 1C and sonication to an average length of 200–700 bp. Chromatin immunoprecipitation was performed using an anti-HDAC1 monoclonal antibody (Upstate Biotechnology). Samples were analyzed by PCR. The following primers for p21 were used: forward, 5′-TGCGTGACAAGAGAATAGCCCAG-3′ and reverse, 5′-TGCAGTTGGCGTCGAGCTGC-3′.

RNA interference

The oligonucleotides used for mouse Id2, HDAC1, and PC1 RNA interference were purchased from Thermo Dharmacon (Lafayette, CO). The oligonucleotides used for mouse c-Myc were purchased from Santa Cruz. The oligonucleotides were transfected using the DharmaFECT siRNA transfection reagent (Dharmacon). Cells were harvested and protein expression was analyzed by western blotting. PC1 expression was also knocked down by the constitutive expression of specific anti-Pkd1 siRNA mediated by a puromycinselectable lentiviral vector, VIRHD/P/siPkd1³²⁹⁷ (kindly provided by Dr G Luca Gusella, Albert Einstein College of Medicine). Mouse inner medullary collecting duct 3 cells were transduced with VIRHD/P/siPkd1³²⁹⁷ or the control VIRHD/P/siLuc lentivector, in which the Pkd1 siRNA was replaced by the anti-luciferase siRNA.²⁴ Following 2 days of puromycin selection (3 days post-transduction), the IMCD/P/siLuc and IMCD/P/siPKD13297 cells were derived for further analysis.

Mouse strain and treatment

To obtain embryos of various genotypes, heterozygous mutant mice were paired and pregnant females were killed on 15.5 d.p.c. to collect embryonic kidneys. For TSA treatment experiments, pregnant Pkd1^{m1Bei +/–} females paired with Pkd1^{m1Bei +/–} males were subcutaneously injected daily, from 8.5 to 14.5 d.p.c., with 0.5 mg TSA (Sigma) per gram mouse body weight or with an equal volume of the vehicle dimethyl sulfoxide. To obtain embryos of Pkd1 and Id2 double knockout, we crossed Pkd1^+/nullId2^+/ females with Pkd1^+/nullId2^+/ males and pregnant females were killed on 15.5 d.p.c. to collect embryonic kidneys. At the end of this treatment, females were killed and embryonic kidneys were collected and fixed in 4% paraformaldehyde. Genomic DNA from the embryos was obtained (XNAT Extract-N-Amp Tissue PCR Kit, Sigma) and genotyped (JumpStart Kit, Sigma).

Statistic analysis

Data are presented as mean±s.e.m. The significance of differences between groups was examined by an unpaired Student's t-test or a one-way ANOVA with Prism 4.0 software (GraphPad Software, San Diego, CA), followed by a Newman–Keuls post hoc test. A P-value <0.05 is considered significant.