Role of FIP200 in cardiac and liver development and its regulation of TNFalpha and TSC-mTOR signaling pathways - PubMed (original) (raw)
Role of FIP200 in cardiac and liver development and its regulation of TNFalpha and TSC-mTOR signaling pathways
Boyi Gan et al. J Cell Biol. 2006.
Abstract
Focal adhesion kinase family interacting protein of 200 kD (FIP200) has been shown to regulate diverse cellular functions such as cell size, proliferation, and migration in vitro. However, the function of FIP200 in vivo has not been investigated. We show that targeted deletion of FIP200 in the mouse led to embryonic death at mid/late gestation associated with heart failure and liver degeneration. We found that FIP200 knockout (KO) embryos show reduced S6 kinase activation and cell size as a result of increased tuberous sclerosis complex function. Furthermore, FIP200 KO embryos exhibited significant apoptosis in heart and liver. Consistent with this, FIP200 KO mouse embryo fibroblasts and liver cells showed increased apoptosis and reduced c-Jun N-terminal kinase phosphorylation in response to tumor necrosis factor (TNF) alpha stimulation, which might be mediated by FIP200 interaction with apoptosis signal-regulating kinase 1 (ASK1) and TNF receptor-associated factor 2 (TRAF2), regulation of TRAF2-ASK1 interaction, and ASK1 phosphorylation. Together, our results reveal that FIP200 functions as a regulatory node to couple two important signaling pathways to regulate cell growth and survival during mouse embryogenesis.
Figures
Figure 1.
Generation of FIP200 KO mice. (A) Schematic representation of the FIP200 targeting vector, mouse FIP200 genomic structure, and the loxP modified FIP200 loci. Large solid triangles represent loxP sites. The relevant restriction sites (B, BglII; N, NcoI), position of probe (thick line) for Southern blotting, and primers (P1, P2, and P3) for PCR genotyping are indicated. Crosses of the mice with targeted allele with EIIa Cre mice result in three possible outcomes at the FIP200 loci (Neo, flox, and Δ allele). (B) Southern blotting analysis of the DNA extracted from ES cells after digestion with corresponding restriction enzymes as indicated. (C) Genomic DNA was extracted from mouse tail and analyzed by PCR (left) and Southern blotting (right), as indicated, to distinguish different types of alleles resulting from Cre-mediated recombination. (D) Western blotting analysis of protein extracts from whole embryos with various genotypes by anti-FIP200 or anti-vinculin, as indicated.
Figure 2.
Defective heart and liver development in FIP200 KO embryos. (A) Gross examination of whole mount FIP200 WT (+/+) and KO (Δ/Δ) embryos at E12.5, E14.5, and E15.5. Note the paleness of FIP200 KO embryos compared with WT littermates at E14.5 and E15.5. (B) Histological sections from the heart of FIP200 WT (+/+) and KO (Δ/Δ) embryos at E14.5. The heart in the KO embryo shows marked left ventricular dilation and a sparsely cellular thin wall that is composed of wisps of thin trabecular myocardium and is devoid of the compact subepicardial myocardium. fw, left ventricular free wall; tm, trabecular myocardium. (C) Histological sections of skin from the dorsum of FIP200 WT (+/+) and KO (Δ/Δ) embryos at E14.5. The marked expansion of the subcutis, the mild expansion of the dermis, and the thin, undulating epidermis are indicative of acute edema. ed, epidermis; d, dermis; sc, subcutis; bf, brown fat. (D) Histological sections of liver FIP200 WT (+/+) and KO (Δ/Δ) embryos at E14.5. Liver from KO embryo showed disrupted architecture with loss of hepatocytes and multifocal variable sized hemorrhages. Also note that many hepatocytes have condensed nuclei (karyopyknosis; red arrow) or fragmented nuclei (karyorrhexis; yellow arrow) or lost their nuclei (karyolysis; black arrow) in the KO embryo (bottom right).
Figure 3.
Reduced S6K activity and cell size in FIP200 KO embryos. (A) Immunohistochemical staining of heart and liver sections from FIP200 WT (+/+) and KO (Δ/Δ) embryos at E12.5 with Ser240/244 phospho-S6 antibody. Note that phospho-S6 staining signal is reduced in these organs in the FIP200 KO embryos. (B) Heart and liver protein extracts prepared from FIP200 WT (+/+) and KO (Δ/Δ) embryos were analyzed by Western blotting using various antibodies, as indicated. Alternatively, they were immunoprecipitated by anti-S6K and analyzed by Western blotting using antibody against Thr389 phospho-S6K and S6K. (C, top) Histograms of mean FSC-H comparing primary FIP200 WT and KO MEFs. (bottom) Relative mean FSC-H + SEM for three independent experiments (results are normalized to FIP200 WT MEFs). *, P < 0.05. (D and E) Heart and liver sections from FIP200 WT (+/+) and KO (Δ/Δ) embryos at E12.5 were stained with WGA-TRITC. (D) Representative fields for each section. (E) Mean + SEM of calculated area of cross sections of cells from three independent experiments in multiple fields. **, P < 0.001.
Figure 4.
Increased apoptosis in FIP200 KO embryos. Immunohistochemical staining of heart (A) and liver (B) sections from FIP200 WT (+/+) and KO (Δ/Δ) embryos at E12.5 or E14.5 with antibody against cleaved caspase-3. There is no immunostaining observed in heart or liver sections from either the KO or WT embryo section at E12.5 or in the WT embryo at E14.5. In contrast, in the KO embryo at E14.5, selected myocardial cells (arrows) show intense cytoplasmic and nuclear staining. Many hepatocytes also show intense cytoplasmic and nuclear staining. In the high-magnification inset, selected hepatocytes are marked with arrows.
Figure 5.
Increased susceptibility of FIP200 KO MEFs to TNFα-induced apoptosis. (A) Primary FIP200 KO and WT MEFs were cultured in DME supplemented with 10% FBS. The cells were left untreated; treated for 1 d with 200 mM sorbitol, 10 μM anisomycin, 5 ng/ml Fas-L, 1 μg/ml TRAIL, and 50 ng/ml TNFα; or cultured in glucose-free medium for 2 d, as indicated. They were then stained with Hoechst to determine the fraction of apoptotic cells, as described in Materials and methods. The mean + SEM from at least three experiments is shown. *, P < 0.001. (B) Primary FIP200 KO and WT MEFs were either left untreated or treated for 1 d with 50 ng/ml TNFα. The fraction of apoptotic cells, necrotic cells, or dead cells (including both apoptotic and necrotic cells) was determined by acridine orange and ethidium bromide costaining. The mean + SEM from at least three experiments is shown. (C) FIP200 KO MEFs were transfected with plasmid encoding HA-tagged FIP200 (fifth and sixth lanes) or empty vector as a control (third and fourth lanes). 1 d after transfection, these and WT MEFs (first and second lanes) were treated with or without 50 ng/ml TNFα for another day, as indicated. Cell lysates were prepared and analyzed by Western blotting using antibodies against cleaved caspase-3, FIP200, and vinculin, as indicated. (D) FIP200 KO MEFs were transfected with empty vector or vector encoding HA-FIP200, along with pEGFP (3:1 ratio). The cells were either left untreated or treated for 1 d with 50 ng/ml TNFα, as indicated. They were then stained with Hoechst to determine the fraction of apoptotic cells in EGFP-positive cells. The mean + SEM from at least three experiments is shown.
Figure 6.
Reduced JNK activation in response to TNFα stimulation in FIP200 KO cells and embryos. (A–C) FIP200 KO and WT MEFs were serum starved overnight. They were then left untreated or treated with 50 ng/ml TNFα for the different periods of time, as indicated. Cell lysates were then analyzed by Western blotting with various antibodies as indicated. (D) FIP200 KO and WT MEFs were infected with recombinant adenoviruses encoding FIP200 (Ad-FIP200) or GFP (Ad-GFP, as control), as indicated. 1 d after infection, cells were serum starved overnight and were left untreated or treated with 50 ng/ml TNFα for 10 min. Cell lysates were analyzed by Western blotting with various antibodies as indicated. (E and F) FIP200 KO MEFs were transfected with empty vector or vector encoding Myc-JNK1, along with pEGFP (3:1 ratio). The cells were either left untreated or treated for 1 d with 50 ng/ml TNFα, as indicated. Cell lysates were prepared and analyzed by Western blotting with various antibodies, as indicated (E). Alternatively, the cells were stained with Hoechst to determine the fraction of apoptotic cells in EGFP-positive populations. The mean + SEM from at least three experiments is shown (F). (G) Western blotting analysis of heart and liver protein extracts from FIP200 WT (+/+) and KO (Δ/Δ) embryos using antibodies against phospho-JNK or JNK, as indicated. (H) Hepatocytes isolated from FIP200 WT (+/+) and KO (Δ/Δ) embryos were serum starved overnight. They were then left untreated or treated with 50 ng/ml TNFα for the different periods of time, as indicated. Cell lysates were then analyzed by Western blotting with phospho-JNK or JNK antibodies, as indicated. (I) FIP200 KO hepatocytes were transfected with empty vector or vector encoding Myc-JNK1, along with pEGFP (3:1 ratio). The cells were either left untreated or treated for 1 d with 50 ng/ml TNFα, as indicated. They were then stained with Hoechst to determine the fraction of apoptotic cells in EGFP-positive populations. The mean + SEM from at least three experiments is shown.
Figure 7.
Association of FIP200 with ASK1 and TRAF2. (A) FIP200 KO and WT MEFs were serum starved overnight. They were then left untreated or treated with 50 ng/ml TNFα for 10 min, as indicated. Cell lysates were then analyzed by Western blotting with various antibodies, as indicated. (B) 293T cells were cotransfected with vector encoding HA-ASK1 or empty vector and plasmid encoding Myc-FIP200 or empty vector, as indicated. Cell lysates were immunoprecipitated with anti-HA or anti-Myc and analyzed by Western blotting with anti-Myc and anti-HA, as indicated. Aliquots of whole cell lysates (WCL) were also analyzed directly by Western blotting with anti-Myc and anti-HA, as indicated. (C) 293T cells were cotransfected with vector encoding Myc-TRAF2 or empty vector and plasmid encoding HA-FIP200. Cell lysates were immunoprecipitated with anti-Myc and analyzed by Western blotting with anti-FIP200 or anti-Myc, as indicated. Aliquots of the lysates were also analyzed directly by Western blotting with anti-FIP200. (D) Lysates from MEFs were immunoprecipitated by anti-ASK1 or anti-TRAF2 or an irrelevant antibody as control, as indicated. They were then analyzed by Western blotting with anti-FIP200, anti-ASK1, or anti-TRAF2, as indicated. Aliquots of the lysates were also analyzed directly.
Figure 8.
Role of FIP200 interaction with TRAF2 and ASK1 in its regulation of TNFα signaling to JNK. (A) Schematic diagram showing the different regions of FIP200 and a summary of their ability to interact with ASK1 or TRAF2. (B and C) 293T cells were cotransfected with vector encoding HA-ASK1 or empty vector and plasmid encoding Myc-CT or -CC (B) or vector encoding Myc-TRAF2 or empty vector and plasmid encoding HA-CT or HA-CC (C), as indicated. Cell lysates were immunoprecipitated with anti-HA or anti-Myc and analyzed by Western blotting with anti-Myc and anti-HA, as indicated. Aliquots of whole cell lysates (WCL) were also analyzed directly by Western blotting with anti-Myc or anti-HA, as indicated. (D) FIP200 KO MEFs were transfected with plasmid encoding HA-tagged FIP200 (fifth and sixth lanes) or empty vector as a control (third and fourth lanes), and WT MEFs were transfected with empty vector. HA-ASK1 was also cotransfected in each transfection. 1 d after transfection, cells were serum starved overnight. They were then left untreated or treated with 50 ng/ml TNFα for 10 min, as indicated. Cell lysates were then immunoprecipitated with anti-TRAF2 and analyzed by Western blotting with anti-ASK1 and anti-TRAF2, as indicated. Aliquots of the lysates were also analyzed by Western blotting with phospho-ASK1 (Thr845), ASK1, or FIP200 antibodies, as indicated. (E) Lysates from cells transfected with HA-CC, HA-CT, HA-N1-859, or empty vector were mixed with lysates from cells cotransfected with HA-ASK1 and Myc-TRAF2. Cell lysates were then immunoprecipitated with anti-Myc and analyzed by Western blotting with anti-Myc and anti-ASK1, as indicated. Aliquots of the lysates were also analyzed directly by Western blotting, as indicated. (F) 293T cells were cotransfected with vectors encoding HA-CC, HA-CT, HA-N1-859, or empty vector and plasmid encoding HA-ASK1 (left) or Myc-JNK1 (right). Cell lysates were then analyzed by Western blotting with various antibodies, as indicated. The phosphorylation levels of ASK1 and JNK are normalized to the expression levels of HA-ASK1 and Myc-JNK, and mean + SEM from three independent experiments are shown on the bottom. (G) MEFs were transfected with empty vector or vector encoding HA-CC, HA-CT, or HA-N1-859, along with pEGFP (3:1 ratio). The cells were either left untreated or treated for 1 d with 50 ng/ml TNFα, as indicated. They were then stained with Hoechst to determine the fraction of apoptotic cells in EGFP-positive cells. The mean + SEM from at least three experiments is shown.
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