Effect Of Tsc1 Knockout On Mouse Mammary Tissue

IntroductionThe genes Tscl and Tsc2 got their names from a severe autosomal dominant disorder, called Tuberous sclerosis complex (TSC), resulting from mutations in one or two of these genes. TSC is characterized by formation of hamartomas in multiple organs and tissues, including derma, smooth muscle, kidney and nervous system. Given the serious consequences of mutations in these genes, the functions of Tsc1 and Tsc2 have been well investigated. The protein hamartin, encoded by Tsc1, and the protein tuberin, encoded by Tsc2, form a functional heterodimer (TSC1/TSC2) with a GTPase-activating protein (GAP) activity, which regulates signaling pathways controlling cell size, cell cycle and cellular proliferation. TSC1 and TSC2 bind to each other via their respective coiled-coil domains. It is TSC2 that contains a C-terminal GAP domain, while binding of TSC1 seems to protect TSC2 against ubiquitin-mediated degradation. Studies have shown that inactivating mutations in either Tscl or Tsc2 give rise to indistinguishable phenotypes in Drosophila. Therefore silence of either of these two genes is equally efficient to inhibit TSC1/TSC2 complex activity.mTORC1, The mammalian target of rapamycin (mTOR), a serine-threonine kinase belonging to the phosphatidylinositol kinase-related kinase family, is a principal downstream target of TSC1/TSC2 complex. mTOR plays its role in two kinds of protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).containing mTOR, regulatory protein of mTOR (Raptor), PRAS40 and mLST8, is directly activated by a small GTPase called Ras-homology enriched in brain (Rheb), whose activation is controlled by TSC1/TSC2 complex. TSC1/TSC2 complex binds to GTP-bound Rheb and stimulates GTP hydrolysis, so as to inhibit the activity of Rheb and subsequently causes inactivation of mTORC1. Therefore loss of Tsc1 or Tsc2 will lead to sustained activation of mTORC1. Activated mTORC1 positively regulates several growth-related cellular process such as transcription and protein translation, which contributes to cell growth and cellular proliferation. Notably, there exist a negative feedback regulation between mTORC1 and AKT, another key protein promoting cell survival and proliferation. When mTORCI is activated, S6K, one of the mTORC1 downstream target, will disrupt the interaction of insulin receptor substrate-1 (IRS-1) with insulin receptors12, leading to a blockage in insulin signaling to AKT, and consequently suppress the AKT activity. Such negative feedback regulation on AKT by mTORC1 surely plays a vital role in controlling proper cell growth and proliferation.Tissue and organ development demands appropriate cell growth, proliferation and apoptosis, where mTORC1 signaling exert a critical role. However embryonic lethality caused by knockout of a vital gene such as Tscl hinder our full knowledge about the role of the gene in development. The mammary gland, unlike other organs, undergoes most of its development postnatally with the onset of puberty rather than in utero15. Here we used a Cre-LoxP conditional knockout strategy to build a mouse model with a specific Tscl deletion in mammary epithelium, which can be born and grow into adult ages and allow us to study the in vivo effects of Tscl deletion in mammary development. Interestingly, Tscl deficiency did not caused hyperplasia of mammary epithelium but evidently retarded the mammary gland branching by decreasing epithelial cell proliferation and increasing cell apoptosis. This study indicated that a modest mTORCI activity was critical for pubertal mammary gland development in mice.Materials and MethodsAnimal. Mice harboring the LoxP-flanked Tscl (Tsc1L/L) allele (129S4/SvJae background) were purchased from Jackson Laboratories (Stock No.005680, Bar Harbor, ME, USA). MMTVCre+ mice were obtained from Center of Model Animal Research at Nanjing University, China. By mating Tsc1L/L mice with MMTVCre+ mice, Tsc1L/wt MMTVCre+ mice were generated, which were then mated with Tsc1L/L mice to generate Tsc1L//LMMTVCre+mice with Tsc1 specifically knockout in mammary epithelia cells. By mating TsclUwt mice with MMTVCre+mice, Tsclwt/wt MMTVCre+mice were generated. All animals were cared for in accordance with guidelines established by the Southern Medical University Animal Care and Use Committee. All experimental protocols were approved by the Southern Medical University Animal Care and Use Committee.Rapamycin treatment.The 4-week-old TsclL/LMMTVCre+mice and Tsclwt/wtMMTVCre+mice were treated with 0.1 mg/kg rapamycin every other day for 2 weeks by i.p. injections. Counterparts given the same amount of saline were taken as control.Whole mount staining, TEBs and branches quantification. The entire 4th mammary gland of each mouse was dissected at 6 weeks of age and spread on a glass slide. Samples were fixed with Carnoy’s fixative for 2-4 hours at room temperature, rinsed in 70% ethanol, and then stained overnight at 4℃ with carmine alum. After dehydration, samples were cleared with xylene and mounted. For TEB quantification, TEBs of greater than or equivalent to 0.03 mm2 were counted under a 40x lens. For branches quantification, the longest primary duct and the secondary duct were counted. Each group took 3 mice to provide the 4th mammary gland, and the TEBs and branches was counted in 3 visual fields per mammary gland under a 40xlens. The average number of each mammary gland was used for statistical analysis and comparison.BrdU incorperation assay and TUNEL assay. Mice at 6 weeks of age were injected i.p. with 10μl/g body weight bromodeoxyuridine (BrdU) 4 hours before sacrifice. The entire 4th mammary gland of each mouse was removed, fixed in 4% paraformaldehyde for 2-4 h at 4℃ and processed to paraffin block. Sections (5μm) were cut, dried at 37℃ overnight and then stored at 4℃ for BrdU and TUNEL analyses. To retrieve nuclear antigens on paraffin-embedded sections, slides were incubated for 20 min in sodium citrate buffer (pH 6.0) at 90 ℃. The sections were blocked with PGB superblock (10% normal goat serum and 10% BSA in 0.5 m PBS) for 2h at room temperature and incubated with monoclonal anti-BrdU antibody (1:1500; Millipore) in PGB diluents (PGB superblock with 1% Triton-X-100) in a humidifier box at 4℃ overnight followed by incubation with fluorescence-conjugated secondary antibodies. To detect apoptotic nuclei, formalin-fixed paraffin-embedded sections were analyzed by TdT digoxigenin nick-end labeling with in situ apoptosis detection kit (Promage) following the manufacturer’s instructions.Immunohistochemistry. Formalin-fixed paraffin-embedded sections were placed in pressure cooker (full pressure for 3min) with appropriate 0.01 M sodium citrate (pH 6.0) for antigen retrieval. Subsequently, sections were incubated in blocking solution consisting of 5% BSA and 10% (v/v) normal goat serum in PBS at room temperature for 1 hour. The primary antibodies TSC1((1:100,abclonal), phosphor-S6 (1:100, CST), phospho-PDK1(1:100, Bioworld), phospho-AKT308 (1:100, Bioworld), phospho-AKT473 (1:100, CST), IRS-1 (1:100, Bioworld), ERα (1:30, Proteintech), cyclin Dl (1:50, Abclonal), cyclin E (1:100, Immunoway), CDK4 (1:100, Abclonal), CDK2 (1:100, Proteintech), p-Rb (1:100, Abclonal) and p27kipl (1:100, Abclonal) were then applied and incubated at 4℃ overnight. Appropriate secondary antibodies were applied for 1 hour. Nuclei were counterstained with hematoxylin.Western Blot. The 4th mammary glands excised from mice were snap frozen in liquid nitrogen, then lysed in cold RIPA buffer (1×PBS,1% NP-40,0.5% sodium dexoycholate,0.1% SDS, protease inhibitor cocktail tablet [Roche] and 1 mM PMSF [Beyotime Biotechnology]). Lysates were incubated on ice for 20 minutes with frequent vortexing and cleared twice by centrifugation (13,200 rpm,10 minutes,4℃). Protein was subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). Membranes were blocked for 60 minutes at room temperature in 5% non-fat milk/Tris-buffered saline/0.1% Tween (TBST). After being washed with TBST, the blots were incubated in primary antibodies for 3 h, which were PS6 (1:2000, CST), S6 (1:2000, CST), TSC1 (1:1000, Proteintech), PDK1(1:1000, ABclonal), phospho-PDK1 (1:1000, Bioworld) phospho-AKT308 (1:1000, Bioworld), phospho-AKT473 (1:1000, CST), AKT1 (1:1000, CST) and β-actin (1:1000, Bioworld). Antibody detection was performed according to the manufacturer’s instructions with ECL Plus Western Blotting Detection System (Genstar, Beijing, China) and developed on film.Statistics. Data were analyzed with unpaired, two-tailed Student’s t-test, using a SPSS 10.0 software. The level of significance was set at P< 0.05.ResultsThe number of branches and TEB bubble of four weeks Tscl-/-mice were less than Tscl+/- mice and Tscl1wt/wt mice, having a statistically significant difference, mammary ductal development of Tsc1-/- mice were retarded, but PS6 activity in mammary epithelial cells of Tsc1-/- mice is stronger than Tsc1+/- mice Tsc1wt.wt^ mice. AKTser308 expression was positive in mammary epithelial cells of Tscl* mice, but in mammary epithelial cells of Tscl+/- mice and Tscl-/- mice, AKTser308 expression was negative.Fourth mammary fat pad of 6-weeks Tsc1-/- mice and Tsc1wt/wt mice were removed for Whole mount staining analysis. The result showed that mammary ductal branches forefront of Tscl-/- mice has not yet passed the lymph nodes, while mammary ductal branches forefront of the control group Tsc1wt/wt mice has passed the lymph nodes, occupying about three-quarters of entire mammary fat pad. statistical analysis about mammary ductal branches and TEB bubble of Tsc1-/- mice and Tsc1wt/wt mice demonstrated that mammary ductal branches and TEB bubble of Tsc1-/ mice were less than the control Tsc1wt/wt mice. the immunohistochemistry and westerm blot analysis about Tscl protein expression level of Tscl-/- mice and Tsc1wt/wt mice showed that Cre-Loxp system indeed played a role in mammary epithelial cells of Tscl-/- mice, TSC1 protein expression level of TSC-/- mice was reduced.brdu experiments showed that the number of BrdU-positive cells of Tsc1-/- mice were less than the control Tsclwt/wt mice,having a statistically significant difference, tunel apoptosis experiment demonstrated that apoptotic cells number of mammary epithelial cell in Tsc1-/-mice were higher than Tsclwt/wt mice,having a statistically significant difference.So far, Conditional knockout of Tscl in mammary epithelium impaired mammary development in mice, contribute to increased apoptosis of mammary epithelial cells and lowered proliferation than Tsc1wt/wt mice4-weeks Tscl-/- mice were treated with rapamycin at a dose of 0.1 mg per kilogram of body weight every other day for 2 weeks by intraperitoneal injection. Control Tsc1-/- mice and Tsc1wt/wtmice were treated with saline at a dose of 0.1 mg per kilogram of body weight every other day for 2 weeks by intraperitoneal injection. Aftern 2 weeks, fourth mammary fat pad of 6-weeks Tsc1-/-mice, Tsclwt/wtmice and Tsc1-/-+rapa were removed for protein level analysis. Analysis about PS6 protein level of Tsc1-/- mice, Tsc1wt/wt mice and Tsc1-/-+rapa mammary tissue showed that rapamycin reduced high PS6 protein level of Tsc-/-mice.rapamycin play a biological role in suppressing activated mTORC1 activity of Tsc1-/-mouse mammary epithelial cells.The Intervention of rapamycin restore the retarded development of mammary ductal branchs of Tsc 1-/-mice, mammary ductal branching and TEB number of Tsc1-/-+rapa mice were higher than only saline-injected control group Tsc1-/- mice and having significant difference.After the treatment of the same dose of rapamycin, the mammary ductal branching and TEB of Tsclwt/wt+rapa mice were less than only saline-injected control group Tsclwt/wtmice, having significant difference, the same dose of rapamycin can induce the supressed mammary ductal development of Tsc1-/- mice, but it inhibited mammary ductal development of Tsclwt/wt mice. Brdu experiments showed that mammary epithelial cell proliferation rate of Tsc1wt/wt+rapa mice are reduced, compared with Tsclwt/wtmice administered with saline, tunel experiments indicated that mammary epithelial cell apoptosis rate of Tsc1wt/wt+rapa mice are increased, compared with Tsc1wt/wt mice administered with saline, having significant difference.For studying the impact of knock out of Rheb gene on the mammary duct of Tsc 1-/- mice, we made a 6-weeks three genotypes of mice:Tsc1-/-Rheb+/+mice、Tsc1-/-Rheb+/- mice and Tsc1-/-’Rheb-/- mice. The result showed that mammary ductal branches forefront of Tsc1-/-Rheb+/+ mice has not yet passed the lymph nodes, mammary ductal branches forefront of Tscl-/- Rheb-/- mice were just just after the front end of the lymph nodes, mammary ductal branches forefront of Tscl-/-Rheb-/-mice not only had passed lymph nodes, its mammary duct had covered the entire 3/4 area of the mammary fat pad.Conditional knockout of Rheb in mammary epithelium of Tsc1-/- mice restored effectively impaired mammary development of Tsc1-/-mice. In summary, rapamycin also reversed effectively impaired mammary development of Tsc1-/- mice. So, TSC1/2→heb→mTORC1→PS6 this signaling pathway play an important role in mammary duct development of mice.Immunohistochemical analysis about mammary fat pad IRS-1 protein of Tsc1-/-mice,Tsc 1-/-rapa mice and Tsc1wt/wt mice showed that nuclear IRS-1 protein of Tsc1-/-mice were down-regulated, compared with the control group Tsc1wt/wt mice, having statistically significant difference. after the intervention of rapamycin, the number of nuclear IRS-1 positive of mammary epithelial cell of Tscl-/-+rapa mice increased, compared with Tsc1-/- mice with statistical significance, constitutively activated PS6 in mammary epithelial cells of Tscl-/- mice down-regulated the number of nuclear IRS-1 protein by negative feedback way.IRS-1 can regulate gene expression of ERa, which was an important regulatory factor during mammary development of mice. ERa and IRS-1 can form a complex in the nucleus, together play a biological role, immunohistochemical analysis about the number of nuclear ERa displayed that ERa positive number of mammary epithelial cells in Tsc1-/- mice reduced,comparing with Tsc1wt/wt mice. after the intervention of rapamycin, the number of nuclear ERa positive of mammary epithelial cell of Tsc1-/- rapa mice increased,compared with Tscl-/- mice with statistical significance. ERa can initiate gene expression SGK3.when SGK3 content decreased, cells tend to undergo apoptosis. By immunohistochemical analysis, we found that, compared to the control group Tsclwt/wt mice, the SGK3 protein content reduced in mammary epithelial cells of Tscl-/- mice.after the rapamycin intervention, the SGK.3 protein content reduced in mammary epithelial cells of Tsc1-/-+rapa mice, decreased SGK3 protein content due to conditional knockout of Tscl in mammary epithelium induce increased apoptosis rate and decreased proliferation rate in mammary epithelial cell. We detected AKTser308 and AKTser473 protein expression in mammary tissue of Tscl-/- mice,Tscl-/-+rapa mice and Tsclwt/wt mice. the result demonstrated that AKTser308 and AKTser473 protein expression of mammary tissue of Tscl-/- mice were lower than Tsc1-/-+rapa mice and Tsc1wt/wt mice. After the rapamycin intervention, AKTser308 and AKTser473 protein expression in mammary tissue of Tsc1-/-rapa mice increased.The detection of p-PDK1 protein content showed that p-PDK1 protein expression of mammary tissue of Tsc1-/- mice were lower than Tscl-/-+rapa mice and Tsc1wt/wt mice After the rapamycin intervention, p-PDK1 protein expression in mammary tissue of Tsc1-/-+rapa mice increased. It also verified suppostion we previously proposed that Conditional knockout of Tscl in mammary epithelium constitutively activated TSC1/2→Rheb→mTORC1→PS6 signaling pathway, the overactivation of PS6 made IRS-1 (insulin receptor substrate-1) protein expression decreased, inhibited the expression of ERa and SGK3, while PI3K-PDK1-AKT as the major signaling pathways signaling pathway downstream of IRS-1 also suppressed. We detected protein expression of cycle-related cytokines, such as:CyclinD1, CyclinE, CDK2, CDK4, pRb, p27 in mammary tissue of Tscl-/- mice,Tsc1-/-+rapa mice and Tsc1wt/wt m immunohistochemical analysis showed that protein expression of cell cycle-related proteins, such as:CyclinD1, CyclinE, CDK2, CDK.4, pRb, p27 of mammary epithelial cells in Tsc1-/- mice decreased, which may lead to cell cycle progression is slower than the control group Tsc1wt/wt mice, the inhibition of mammary duct development of Tsc1-/- mice, aftern rapamycin intervention, protein expression of cell cycle-related proteins, such as:CyclinD1, CyclinE, CDK2, CDK.4, pRb, p27 of mammary epithelial cells in Tsc1-/=+rapa mice increased.ConclusionsThis study supports the notion that a balanced mTORC 1 activity was critical for pubertal mammary gland development in mice. Loss of Tscl and consequently sustained activation of mTORC 1 results in impaired pubertal mammary gland development. The specific mTORC 1 inhibitor rapamycin used at a dose of 0.1 mg per kilogram of body weight every other day for 2 weeks can restore the mammary development of the Tsc1 L/LLMMTVCre+mice, whereas delayed the mammary development of the Tsc 1 wt/wtMMTVCre+counterparts. Mechanisms underlying the impaired mammary development induced by Tscl deficiency may involve:1, suppression of AKT by sustained activation of mTORC1; 2, inhibition on nuclear ERa signaling; and 3, down-regulation of Cyclin Dl, Cylin E, CDK2 and CDK4, which hold back the cell cycle progression. We speculate that down-regulation of nuclear ERa, Cyclin D1, Cyclin E, CDK2 and CDK4 are related to depressed AKT activity. The negative feed back regulation on AKT by activated mTORC1 probably plays a key role in the retarded mammary development induced by Tsc1 deficiency.