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, 24 (1), 407-19

Essential Role of STAT3 in Postnatal Survival and Growth Revealed by Mice Lacking STAT3 Serine 727 Phosphorylation

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Essential Role of STAT3 in Postnatal Survival and Growth Revealed by Mice Lacking STAT3 Serine 727 Phosphorylation

Yuhong Shen et al. Mol Cell Biol.

Abstract

A large number of extracellular polypeptides bound to their cognate receptors activate the transcription factor STAT3 by phosphorylation of tyrosine 705. Supplemental activation occurs when serine 727 is also phosphorylated. STAT3 deletion in mice leads to embryonic lethality. We have produced mice with alanine substituted for serine 727 in STAT3 (the SA allele) to examine the function of serine 727 phosphorylation in vivo. Embryonic fibroblasts from SA/SA mice had approximately 50% of the transcriptional response of wild-type cells. However, SA/SA mice were viable and grossly normal. STAT3 wild-type/null (+/-) animals were also normal and were interbred with SA/SA mice to study SA/- mice. The SA/- mice progressed through gestation, showing 10 to 15% reduced birth weight, three-fourths died soon after birth, and the SA/- survivors reached only 50 to 60% of normal size at 1 week of age. The lethality and decreased growth were accompanied by altered insulin-like growth factor 1 (IGF-1) levels in serum, establishing a role for the STAT3 serine phosphorylation acting through IGF-1 in embryonic and perinatal growth. The SA/- survivors have decreased thymocyte number associated with increased apoptosis, but unexpectedly normal STAT3-dependent liver acute phase response. These animals offer the opportunity to study defined reductions in the transcriptional capacity of a widely used signaling pathway.

Figures

FIG. 1.
FIG. 1.
Generation of the Stat3 S727A mutant mice. (A) Schematic diagrams of the mouse endogenous wild-type Stat3 allele, S727A targeting vector, and the resulting S727A mutant allele after homologous recombination. Exons are shown as rectangles. Primers a1 and a2 were matched to wild-type serine 727 or mutated alanine sequences, respectively, and were used in pairs with primer b to perform PCR screening for mutation. Primers b and c were used to PCR amplify the fragment in between, which was sequenced to confirm the S727A mutation. N, Neo cassette; A(n), polyadenylation sequence; L, loxP sequence. (B) Southern blot analysis of EcoRI-digested genomic DNA from +/+, SA/+, and SA/SA mice using the probe shown in panel A. The probe detects a 14.5-kb band from the wild-type allele and a 6.5-kb band from the mutant S727A allele. (C) PCR analysis of the genomic DNA analyzed in panel B. Primers a1 and b detect the ∼4.5-kb band from the +/+ allele, and primers a2 and b detect the ∼6.5-kb band from the mutant S727A allele. (D) Western blot analysis to check serine phosphorylation. Whole-cell extracts prepared from +/+, SA/SA, or −/− MEFs untreated or treated with OSM for 30 min were immunoprecipitated with an anti-STAT3 antibody, followed by Western blotting using an antibody against phospho-STAT3 (Ser727). The same extracts were also straightly probed with an anti-phospho-STAT3 (Tyr705) antibody or stripped and reprobed with an anti-STAT3 antibody.
FIG. 2.
FIG. 2.
The STAT3SA mutation decreases STAT3 transactivation activity in MEFs. (A) MEFs were prepared from E13.5 STAT3+/+, STAT3+/SA, and STAT3SA/SA littermate embryos. MEFs were transiently transfected with an M67 luciferase reporter that contains four copies of the STAT binding site in its promoter, left untreated (no), or treated with IL-6 and IL-6R (IL-6) for 6 h and then harvested for a luciferase assay. Shown are average results from two, three, and three lines of MEFs derived from STAT3 +/+, +/SA, and SA/SA animals, respectively (triplet samples were tested for each cell line). (B) As for panel A, MEFs were prepared from STAT3+/+, +/−, and two SA/− littermate embryos. Transiently transfected MEFs were left untreated (no) or treated with IL-6 and IL-6R (IL-6) or OSM for 6 h. (C) As for panel B, transfections were done in STAT3+/− or −/− MEFs. (D) MEFs prepared from STAT3+/+, +/−, and two SA/− littermate embryos were treated with OSM (25 ng/ml) or IFN-γ (10 ng/ml) for the indicated times, and the indicated gene expression was analyzed by RT-PCR. For the OSM treatment, SOCS3, Fos, and JunB expression was also quantified and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by phosphorimager analysis; data are shown in the graphs underneath (SOCS3/GAPDH, Fos/GAPDH, and JunB/GAPDH).
FIG. 2.
FIG. 2.
The STAT3SA mutation decreases STAT3 transactivation activity in MEFs. (A) MEFs were prepared from E13.5 STAT3+/+, STAT3+/SA, and STAT3SA/SA littermate embryos. MEFs were transiently transfected with an M67 luciferase reporter that contains four copies of the STAT binding site in its promoter, left untreated (no), or treated with IL-6 and IL-6R (IL-6) for 6 h and then harvested for a luciferase assay. Shown are average results from two, three, and three lines of MEFs derived from STAT3 +/+, +/SA, and SA/SA animals, respectively (triplet samples were tested for each cell line). (B) As for panel A, MEFs were prepared from STAT3+/+, +/−, and two SA/− littermate embryos. Transiently transfected MEFs were left untreated (no) or treated with IL-6 and IL-6R (IL-6) or OSM for 6 h. (C) As for panel B, transfections were done in STAT3+/− or −/− MEFs. (D) MEFs prepared from STAT3+/+, +/−, and two SA/− littermate embryos were treated with OSM (25 ng/ml) or IFN-γ (10 ng/ml) for the indicated times, and the indicated gene expression was analyzed by RT-PCR. For the OSM treatment, SOCS3, Fos, and JunB expression was also quantified and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by phosphorimager analysis; data are shown in the graphs underneath (SOCS3/GAPDH, Fos/GAPDH, and JunB/GAPDH).
FIG.3.
FIG.3.
Growth retardation of STAT3SA/− mice. (A) Representative male littermates at day P24. The mouse on top is SA/−, and the one on the bottom is SA/+. (B) Growth curves with standard deviations of STAT3+/SA (filled squares; total of 12 animals) and STAT3SA/− (filled circles; total of 8 animals) male mice from day P3 to 8 weeks of age. P was <0.0001 in the 2 to 6 weeks. (C) Representative female littermates at day P24. The top animal is SA/−, and the one on the bottom is SA/+. (D) Growth curves with standard deviations of STAT3+/SA (filled squares; total of 13 animals) and STAT3SA/− (filled circles; total of 6 animals) female mice from day P3 to 8 weeks of age. P was <0.0001. (E) A litter of E18.5 embryos showing reduced weight of SA/− mice during embryogenesis. The ones marked with an M are SA/−, and the rest are SA/+. (F) Weight of STAT3+/SA (11 pups) and STAT3SA/− (13 pups) embryos at E18.5. Three asterisks indicate that P was <0.001. (F) Body and tail lengths of STAT3+/SA and STAT3SA/− mice at 24 days of age. Top, males; bottom, females. Filled bar, SA/+; open bar, SA/−. Three asterisks indicate that P was <0.001. (G) Organ weights of STAT3+/SA (total of six animals) and STAT3SA/− (total of four animals) male mice at 24 days of age. The weight of each organ of the SA/+ mice was assigned as 100%. Filled bar, SA/+; open bar, SA/−. For liver and spleen, P < 0.01; for the rest, P < 0.001. (F) As for panel G but for female mice. Total of five SA/+ and six SA/− mice. Spleen, P = 0.016; for the rest, P < 0.0001.
FIG. 4.
FIG. 4.
Altered IGF-1 levels in STAT3SA/− mice. (A) Serum IGF-1 level of 24-day-old littermate SA/+ and SA/− males and females. Four to six mice of each genotype were analyzed. Filled bar, +/SA; open bar, SA/−. Three asterisks indicate that P was <0.001. (B) Serum IGF-1 levels and weight of 8-day-old littermate SA/+ and SA/− mice. Filled bar, +/SA; open bar, SA/−. SA/+, n = 8; SA/−, n = 4. Three asterisks indicate that P was <0.001. (C) Serum IGF-1 level and weight of SA/+ and SA/− newborns. SA/+, total of 21 pups; SA/−, total of 22 pups; SA/− High, 16 SA/− pups grouped according to higher IGF-1 levels; SA/− Low, 6 SA/− pups grouped according to lower IGF-1 levels. Two asterisks indicate that P was <0.01; three asterisks indicate that P was <0.001.
FIG. 5.
FIG. 5.
GH pathway in SA/− mice. (A) Serum GH levels in 24-day-old males and females at the time of sacrifice. Filled bar, SA/+; open bar, SA/−. P = 0.011 (one asterisk indicates that P was <0.05). (B) MUP levels in SA/+ and SA/− mice. Urine was collected from 24-day-old littermate animals at the time of sacrifice and checked by SDS-15% PAGE. Samples from the SA/− animals were loaded adjacent to those from the littermate SA/+ animals. The MUP is indicated by the arrow.
FIG. 6.
FIG. 6.
Decreased thymocyte survival in STAT3SA/− mice. (A) Thymocyte numbers of littermate STAT3+/SA and STAT3SA/− mice at ages of 7 days (three mice each), 10 weeks (four mice each), and 6 months (five mice each). The values shown are the mean cell number per thymus with the standard error. Two asterisks indicate that P was <0.01. (B) Thymocyte apoptosis of the littermate STAT3+/SA and STAT3SA/− mice. Thymocytes from the mice examined in panel A were stained with fluorescein isothiocyanate-Annexin V and propidium iodide; the mean percentages of the Annexin V-positive cells are shown with standard errors. One asterisk indicates that P was <0.05; two asterisks indicate that P was <0.01.
FIG. 7.
FIG. 7.
Induction of liver acute phase response gene expression in STAT3SA/− mice. Littermate STAT3+/+ and STAT3SA/− mice were injected with turpentine subcutaneously or recombinant IL-6 intraperitoneally. After 12 h of turpentine injection or 6 h of IL-6 injection, liver total RNA was prepared from each animal and analyzed by RT-PCR. As negative controls, mice were either left untreated or injected with sterile pyrogen-free saline. SAA, serum amyloids A; SAP, serum amyloids P; FB, fibrinogen; HP, haptoglobin; HPX, hemopexin; AGP, α1-acid glycoprotein.

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