Leukemia inhibitory factor inhibits neuronal terminal differentiation through STAT3 activation

Abstract

The discovery of stem cells in the adult central nervous system raises questions concerning the neurotrophic factors that regulate postnatal neuronal development. Olfactory receptor neurons (ORNs) are a useful model, because they are capable of robust neurogenesis throughout adulthood. We have investigated the role of leukemia inhibitory factor (LIF) in postnatal neuronal development by using ORNs as a model. LIF is a multifunctional cytokine implicated in various aspects of neuronal development, including phenotype determination, survival, and in response to nerve injury. LIF-deficient mice display significant increases, both in the absolute amount and in the number of cells expressing olfactory marker protein, a marker of mature ORNs. The maturation of ORNs was significantly inhibited by LIF in vitro. LIF activated the STAT3 pathway in ORNs, and transfection of ORNs with a dominant negative form of STAT3 abolished the effect of LIF. These findings demonstrate that LIF negatively regulates ORN maturation via the STAT3 pathway. Thus, LIF plays a critical role in controlling the transition of ORNs to maturity. Consequently, a population of ORNs is maintained in an immature state to facilitate the rapid repopulation of the olfactory epithelium with mature neurons during normal cell turnover or after injury.

Figure 1

Expression of OMP, ACIII, and O/E-1 in olfactory epithelium. (a–d) Immunoreactivity was assessed in WT (a, c, and e) and LIF-deficient mice (b, d, and f) for OMP (a and b), ACIII (c and d), and O/E-1 (e and f). Ax, axon bundle; BV, blood vessel; OE, olfactory epithelium; LP, lamina propria; SCL, sustentacular cell layer. The dotted line represents the basal lamina. (g) Expression of OMP and ACIII in WT (+/+) and LIF-null mice (−/−) was assessed by immunoblot. Equal loading of the protein samples was confirmed by probing for monomeric actin. (h) Summary of OMP and O/E-1 staining in olfactory epithelium. Cells in areas of 0.04 mm² (0.2 × 0.2 mm; width × height) were counted. Data represent the mean ± SEM of cell numbers counted in at least 10 different areas from three independent experiments. Statistical significance is indicated as *** (P < 0.0001, Student's t test).

Figure 2

LIF expression in olfactory epithelium. (a–f) In situ hybridization analysis of LIF in olfactory epithelium. Expression of LIF mRNA was determined in coronal sections of adult mouse nasal cavity by using antisense LIF riboprobe (a and b). To ascertain the specificity of the probe, sense controls were also performed (c and d). Expression of OMP in olfactory epithelium was also examined by using antisense and sense OMP riboprobe (e and f, respectively). (g) Immunoblot analyses of LIF signaling components in olfactory tissues. The molecular mass (in kDa) is indicated at right.

Figure 3

LIF induced activation of STAT3 in ORNs. (a) Western blot analysis of LIF-induced STAT3 activation. Tyrosyl phosphorylated STAT3 was detected by using an anti-phosphoSTAT3 antibody. Primary cultures of ORNs were treated with or without LIF (10 ng/ml) for 15 min. K252a (100 nM) was added before stimulation for 30 min. (b) Immunocytochemical analysis of the activation of STAT3 with LIF addition. Primary cultures of ORNs were treated with LIF (10 ng/ml) for 15 min. After fixation, immunocytochemistry was performed using anti-phospho-STAT3 antibodies. (c) Immunofluorescence analysis of the translocation of STAT3 into the nucleus on LIF stimulation. Primary cultures of ORNs were treated with LIF (10 ng/ml) for 15 min. After fixation, immunofluorescence was performed with anti-phospho-STAT3 antibodies. The translocation of STAT3 into the nucleus was visualized by using rhodamine-conjugated antibodies and CyQUANT GR dye. STAT3 proteins in the nucleus appear yellow. (d) Electrophoretic mobility shift assay. LIF-induced DNA binding activity of STAT3. * indicates SIE-STAT complex. Primary cultures of ORNs were incubated for 15 min as described above. K252a was preincubated for 15 min before stimulation. As a control, no cell lysate was added. The isolated nucleus fractions were incubated with radioactive-labeled SIE and subjected to 4% polyacrylamide, nondenaturing gel electrophoresis. For antibody supershift assay, SIE-STAT3 complex was incubated with anti-STAT3 antibodies (1 μg) for 15 min. ** indicates the supershifted SIE–STAT complex.

Figure 4

Transfection of dominant negative STAT3 into ORNs. Transfection of dominant negative STAT3 abolished the effect of LIF on OMP expression of ORNs. a-l. Primary cultures of ORNs were transfected with vectors containing dominant negative STAT3. Transfection of vectors was confirmed by expression of GFP. OMP and GFP were visualized by immunofluorescence. Immunofluorescence was assessed in ORNs transfected with control vector (pEGFP) (a–f) and with dominant negative STAT3 vector (pEGFP-D/N STAT3) (g–l). ORNs were incubated in medium containing NGF either without (a–c and g–i) or with LIF (d–f, j–l). Expression of OMP and GFP was visualized by FITC- and rhodamine-conjugated antibodies respectively, thus, coexpression is visualized as yellow. (m) Summary of the effect of dominant negative STAT3 on OMP expression in ORNs. OMP expression in ORNs was significantly decreased in medium containing LIF (100 ng/ml) than in medium containing NGF (25 ng/ml) alone (P < 0.0001, Student's t test). The effect of LIF was abolished by transfection of dominant negative STAT3 in ORNs (P = 0.0004, Student's t test). Cell numbers were counted from three independent experiments. Data represent mean ± SEM.

Figure 5

STAT3 binding to OMP promoter. (a) Summary of transcription factor binding sites in OMP promoter region. (b) Competitive binding assay. (See text for details.)