PMC

LIF activates Stat3 but not other Stats in LE. (A) Nuclear extracts were prepared from day-4 LE with (+) or without (−) LIF treatment. ³²P-labeled DNA oligomers (CS Oligo) for the different consensus Stat-binding sites were used to assay the DNA-binding activities, revealing the specific activation of Stat3. (B) Stat3-specific binding activity was confirmed by it being out-competed with 100-fold unlabeled Stat3, but not by Stat1 oligomers and (C) antibody to Stat3 generated a supershifted band (arrow). Rb, rabbit Ab; ST, Stat Ab.

LE is a LIF target tissue in the uterus. (A) In situ hybridization and immunostaining localizes LIFRα mRNA and protein to the LE and gp130 mRNA and protein predominantly in GE (arrows). (B) Analysis of purified LE by ribonuclease protection assay identifies LIFRα and gp130 mRNA transcripts and (C) multiple transcripts detected by Northern blot. (D) LIF receptor (190-kDa) and gp130 (125-kDa) protein are detected by Western blot analysis of LE protein extracts. The purity of LE preparation was confirmed by the absence of the LIF message that is expressed in the GE and the presence the prostaglandin E2 receptor message, which is specific for the LE (B). RPL19, ribosomal protein L19.

Nuclear translocation of Stat3 is attenuated in LIF −/− mice. (A) Stat3 immunoreactivity in uterine sections reveals a uniform nuclear localization of Stat3 to the entire LE on day 4 in +/+ but not −/− mice. (B) Higher-powered views are shown for days 3–5 of Stat3 localization to the LE and GE nuclei. (C) A summary table of the stained uterine sections is shown where the number of uteri showing predominant cytoplasmic localization (c > n) of Stat3 or nuclear localization (c < n) is presented in both the LE and GE. A few day-3 +/+ sections showed occasional nuclear translocation of Stat3 in both LE and GE. In day-3 LIF −/−, no translocation was evident. In D4 +/+, more than half of the uteri showed complete nuclear localization of Stat3 in the entire LE but not in GE. In contrast, LIF −/− uteri showed little or no nuclear localization in the LE. In day-5 +/+ animals, the nuclear localization of Stat3 in LE is absent in the LE between implantation sites with some nuclear staining detected in GE. LIF −/− mice show a marked reduction and absence of Stat3 nuclear location in both LE and GE. WT, wild type.

LIF activation of Stat3 in LE is regulated temporally. Purified LE was treated with LIF and the total p-Tyr-Stat3 and Stat3 protein levels determined by Stat3 phospho-specific and Stat3 antibodies on the same blot. (A) Both LIF and HIL-6 fusion protein (150 ng/ml) increase p-Tyr-Stat3 levels. Increased Stat3 phosphorylation after LIF treatment occurs in LE from day-4 LE, but not from days 3 or 5. (B) Such temporal restriction in activation is observed for LIF but not EGF. EGF (100 ng/ml) activates MAPK by a 30–60% increase as shown by the ratios of phosphorylated MAPK to MAPK (the numbers under each lane are the ratios of P-MAPK to MAPK). (C) Analysis of the temporal differences of LIF activation are shown with the ratios of p-Tyr-Stat3 to total Stat3 being higher in day-4 LIF-treated LE (n = 4); this is statistically significant (*, P < 0.035, Student's t test) from that of day-4 untreated LE (n = 4). LIF did not induce Stat3 activation before or after day 4 (n = 4 for day 3 and n = 3 for day 5), revealing that responsiveness of the LE to LIF is regulated temporally.

LIF binding to the LE remains unchanged. Scatchard analysis of ¹²⁵I-labeled LIF binding to LE cells on days 3 (⧫), 4 (■), and 5 (●). Specific binding was calculated by subtracting radioactivity bound in the presence of excess unlabeled LIF (nonspecific binding) from total binding. Points are the result of five separate experiments. Optimal (weighted least squares) estimates of affinity constants (K values), dissociation constants (K_d), and binding capacities (R) were analyzed by the LIGAND program (). Alternative models were tested to determine the existence of multiple binding sites with the one-binding-site model providing the most parsimonious fit. Binding parameters for data corresponding to days 3, 4, and 5 were compared and found not to differ statistically. Apparent dissociation constants were as indicated, and binding capacities (K_d) for days 3, 4, and 5 were 63.06, 87.25, and 87.26 pM, respectively. F9 EC cells were used as a positive control as described (). B/F, bound/free.