Tetratricopeptide repeat domain 9A negatively regulates estrogen receptor alpha activity

Abstract

Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.

Keywords: Co-chaperone.; ERα; Knockout Mouse; Mammary gland; TTC9A.

Figure 1

Ttc9a transgene construct and screening strategy. (A) Strategy for targeted disruption of Ttc9a gene. Ttc9a exon1 was replaced by neomycin cassette by homologous recombination as indicated by the dotted lines in the mouse ES cells. Exons are indicated by filled boxes; homology arms are indicated by thick lines; introns are indicated by thin lines; and loxp sites are indicated by triangles. (B) Southern blot analysis of Sca I-digested ES cell and tail genomic DNA from wild (+/+), heterozygote (+/-) and homozygous (-/-) Ttc9a KO mice. 5' probe detect a ~11 kb wild-type (Wt) and a ~4 kb mutant (Mut) band. (C) Genotyping tail DNA by PCR screening. The wild-type allele yielded a 200 bp band size and the mutant allele gave 654 bp. (D) TTC9A protein expression is abolished in Ttc9a^-/- mice. Total protein extract from mice tissues (brain, mammary gland (Mg), lung and uterus) were analysed by immunoblotting with TTC9A specific antibody. GAPDH was used as a loading control. The blot shows complete absence of TTC9A protein in the 4 tissues assayed from Ttc9a^-/- mice.

Figure 2

Ttc9a KO mice show greater body, spleen and thymus mass than wild-type mice. (A) Both male and female Ttc9a^-/- mice at postnatal day 2 showed greater body weight than Ttc9a^+/+ mice. (B) Body weights of male mice from 4-12 week old were similar across Ttc9a^+/+, Ttc9a^+/- and Ttc9a^-/- mice. (C) The body weights of Ttc9a^-/- female mice was significantly higher than Ttc9a^+/+ mice at 6- and 10-week of age. (D) The spleen and (E) thymus mass in the Ttc9a^-/-mice were significantly greater compared to Ttc9a^+/+ mice. Animal and organ weight data are expressed as means ± SEM; *p value < 0.05; **p value < 0.01. Supplement data indicates the number of animals used in each analysis.

Figure 3

Ttc9a KO mice exhibit increased mammary ductal branching. Whole-mount images of inguinal mammary gland from Ttc9a^+/+, Ttc9a^+/- and Ttc9a^-/- mice at 6 week old at (A) 4X magnification and (B) 10X magnification. Morphometric analysis using Image J quantitated the ductal development in the three genotypes. There was significant increase in the (C) ductal branch points in Ttc9a^-/- mice compared to Ttc9a^+/+, but no difference in the (D) ductal length. (E) Ttc9a^-/- and Ttc9a^+/-mammary gland showed an increase of TEBs compared to Ttc9a^+/ but this difference was not statistically significant. LN - lymph node; TEB - terminal end bud. Data are expressed as means ± SEM; * p < 0.05.Supplement data indicates the number of animals used in each analysis.

Figure 4

The mammary glands from Ttc9a KO mice are more responsive to estradiol. Representative whole mount images of the 4^th inguinal mammary gland of (A) control (ctrl) (B) 17β estradiol benzoate (E2) and (C) 17β estradiol benzoate and progesterone (E2P) treated Ttc9a^+/+and Ttc9a^-/- mice. The mammary ductal morphology between the Ttc9a^+/+ and Ttc9a^-/- gland was quantified using the Image J software. The histograms illustrated significant increases in (D) ductal branch points and (E) ductal length in Ttc9a^-/- mice compared to Ttc9a^+/+ on E2P and E2 treatment respectively. Also there was a significant increase in the (F) TEB count in Ttc9a^-/- gland on E2 treatment compared to Ttc9a^+/+. This increased ductal morphology were associated with greater induction of E2 target genes (PR, Areg, Greb1 and Muc1) in Ttc9a^-/- mice than Ttc9a^+/+ mice as analyzed by real time PCR (G-J). Data are expressed as means ± SEM; for each sample set. *p<0.05, **p< 0.01.Supplement data indicates the number of animals used in each analysis.

Figure 5

TTC9A down-regulates ERα transcriptional activity in breast cancer cells. (A, B) ERE-luciferase assay showing a significant decrease in ERα transcriptional activity by TTC9A (25ng) and up-regulation in ERα transcriptional activity with TTC9A siRNA knockdown in MCF-7. Western blot showing that endogenous ERα expression level was not affected by TTC9A over-expression, and TTC9A protein level was drastically reduced by knockdown. Result shown (mean±SEM (n = 3) is representative of two independent experiments. The p values were obtained by Student's t-test. **p<0.01. (C) ERE-luciferase assay showing the effects of FKBP38 and FKBP51 (50ng) on ERα's transcriptional activity in MCF-7 cells. Western blot showing that endogenous ERα expression level was not affected by FKBP over-expression. Luciferase reporter assay result (mean±SEM (n = 3) shown is representative of two independent experiments *p<0.05, **p<0.01. (D) FKBP38 and FKBP51 interact with ERα and Hsp90. COS7 cells were transfected with ERα alone or together with FLAG-FKBP38 or FLAG-FKBP51. Anti-FLAG antibody pulled down both FKBPs and co-immunoprecipitated both ERα and Hsp90. ERα and Hsp90 were detected using anti-ERα and anti-Hsp90 antibodies respectively. FKBP38 and 51 were detected by anti-FLAG antibody. (E) TTC9A does not interact with ERα and Hsp90. COS7 cells were transfected with pXJ-FLAG control vector with ERα or FLAG-TTC9 with ERα. Anti-FLAG antibody pulled down FLAG-TTC9, but no ERα or endogenous Hsp90 proteins were co-immunoprecipitated. (F) TTC9A interacts with FKBP38 and FKBP51. COS7 cells were transfected with GFP-TTC9A and FLAG-FKBP38 or FLAG-FKBP51. Anti-FLAG antibody pulled down both FKBPs and co-immunoprecipitated TTC9A and Hsp90 at the same time. (G) TTC9A interacts with endogenous FKBP51. HeLa cells were transfected with FLAG-TTC9A or control vector and treated with 10 nM dexamethasone to induce the endogenous FKBP51. Anti-FLAG antibody was used to pull-down FLAG-TTC9A in the whole cell lysates and the co-immunoprecipitated FKBP51 was detected by anti-FKBP51 antibody.

Figure 6

The protein sequence of the Ttc9 family is conserved at the regions corresponding to the TPR domains of Ttc9a. (A) The TTC9A protein comprises of three TPR domains. (B) Alignment of the mouse TTC9A (222 aa), TTC9B (239 aa) and TTC9C (171 aa) amino acids using Vector NTI software. Yellow - Identical amino acid residues; Green - residues of similar properties; Blue - conserved residues across species.