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PNAS 102 (26): 9188-9193

Copyright © 2005 by the National Academy of Sciences.

Insights into regulation of human Schwann cell proliferation by Erk1/2 via a MEK-independent and p56Lck-dependent pathway from leprosy bacilli

Nikos Tapinos, and Anura Rambukkana *

Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, Bronk Building Room 501, 1230 York Avenue, New York, NY 10021

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Fig. 1. Intracellular M. leprae promote Schwann cell cycle progression and proliferation. (A-C) Intracellular localization of M. leprae (ML) in purified primary human Schwann cells after 15 (A) and 30 (B) days of infection as determined by immunofluorescence and electron microscopy. In A, Schwann cell marker S-100 is red, and M. leprae-specific PGL-1 is green (indicated by the arrows). B shows electron microscopy. (C) Quantification of Schwann cells harboring intracellular M. leprae. **, P < 0.001, Student's t test. (D) BrdUrd uptake of M. leprae-infected (+ML) and control (-ML) Schwann cells. (E) Division of cell nuclei of infected cells as determined by DAPI labeling (Insets). Cells are double-stained with antibodies to ErbB2 (green) and PGL-1 (red/yellow). Asterisks in Insets show the dividing nuclei (arrows). (F) Cell cycle distribution of asynchronously growing M. leprae-infected human Schwann cells as analyzed by FACScan using propidium iodide. The percentage of total cells in G1, S, and G2 phases of the cell cycle are shown. The data are from three individual experiments from Schwann cells purified from two different organ donors. *, P < 0.005; **, P < 0.001, Student's t test. (G) Total Schwann cell counts from asynchronized cultures before (day 0; starting with equal cell numbers) and 7, 15, and 30 days after M. leprae infection are shown from four individual experiments. **, P < 0.0005, Student's t test.


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Fig. 2. Intracellular M. leprae induce cyclin D1 gene and protein expression in human Schwann cells; role in G1/S-phase progression is shown. (A and B) Fold increase of differentially expressed cyclin D1 and p21 genes in M. leprae-infected Schwann cells as analyzed by using Affymetrix human GeneChips. (A) Differentially expressed cyclin D1 and p21 genes in asynchronized Schwann cells at postinfection day 3, 7, and 30. (B) Cyclin D1 and p21 gene expression in 30-day-infected Schwann cells from three individual experiments (experiments are labeled 1-3) (P < 0.001). (C) Quantitative real-time PCR analysis of cyclin D1 and p21 genes in human Schwann cells 7 and 30 days after M. leprae infection. Differentially expressed genes are presented after normalization with GAPDH gene expression relative to control expression. (D) Protein levels of cyclin D1 and p21 and phosphorylation of Erk1/2 from asynchronized cultures of human primary Schwann cells infected with M. leprae. Expression was measured by Western blot analysis of total cell extracts using antibodies to cyclin D1, p21, and phospho-specific (p) Erk1/2. (E) Overexpression of the T156A cyclin D1 DNM decreased the G1 phase progression of M. leprae-infected Schwann cells. Cell cycle FACS analysis of 30-day-infected human primary Schwann cells transfected with vector alone and T156A/cyclin D1 DNM. Data are from three independent cell cycle FACS experiments showing the mean percentage of cell population in G1, S, and G2 phases. *, P < 0.005, Student's t test.


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Fig. 3. Role of MEK-independent Erk1/2 signaling in M. leprae-induced cyclin D1 and G1/S-phase progression. (A-C) Intracellular M. leprae activate Erk1/2 by means of MEK/PI3K-independent pathways. (A) Total lysates of 30-day-infected human Schwann cells purified from two different organ donors were synchronized for 48 h and then blotted with anti-phospho-Erk1/2 antibody. (B) Thirty-day-infected and control cells were synchronized for 48 h in the continuous presence of MEKI UO126 and PI3KI LY294002, and SOS inhibitory peptide (SOSI) and cell lysates were blotted with antibody to phospho-Erk1/2 and phospho-MEK1/2. (C) Active phospho-Erk1/2 was immunoprecipitated from lysates of synchronized human Schwann cells by anti-phospho Erk1/2 antibody, and the resulting immunoprecipitate was then incubated with a fusion protein of Elk-1 transcription factor. Phosphorylation of Elk-1 at Ser-383, a major phosphorylation site of Erk1/2, was determined by phosho-Elk-1 antibody as a measure of kinase activity. (D) DNM of Erk2 inhibits M. leprae-induced S-phase entry. Infected human Schwann cells were transfected with Erk2/p44mapk DNM T192A and vector alone, and cell cycle kinetics were analyzed by FACS. *, P < 0.0001, Student's t test. (Inset) Cyclin D1 expression in transfected infected cells with vector alone (lane 1) and DNM T192A (lane 2) are shown. In all experiments, {beta}-actin labeling is shown to indicate equal amount of proteins in each lane.


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Fig. 4. M. leprae-induced Erk1/2 phosphorylation is mediated by p56Lck by means of a PKC{epsilon}-dependent and MEK-independent pathway. (A) Thirty-day-infected Schwann cells were serum-starved for 48 h in the continuous presence of PI3KI and MEKI with or without PKCI bisindolylmaleimide. Erk1/2 and MEK1/2 phosphorylation was analyzed by immunoblotting with phospho-specific antibodies to Erk1/2 and MEK1/2, respectively. Protein loading was controlled by {beta}-actin expression in all experiments. (B and C) Transient transfection of PKC{epsilon}-DNM abrogated M. leprae-induced phosphorylation of Erk1/2. (B) Indicated PKC-DNM constructs were expressed in 30-day-infected Schwann cells and analyzed by Western blotting using antibody to phospho-Erk1/2. (C) Induction of M. leprae-induced phosphorylation of PKC{epsilon} and its inhibition by PKCI. (D) LckI abrogated M. leprae-induced Erk1/2 activation in the presence of MEKI and PI3KI. Western blot analysis of infected cells incubated with MEKI and PI3KI in the absence and presence of LckI. (E) Expression of Lck in human primary Schwann cells (control and infected cells) was analyzed by RT-PCR ({beta}-actin was used as a housekeeping gene). (F)(Left) Thirty-day-infected cells and controls from two individual donors (#1 and #2) were synchronized for 48 h, and the lysates were analyzed by Western blot using antibody to total Lck. (Right) Antibody activity to protein extract of human sciatic nerve is shown. (G) M. leprae-infected cells were incubated with LckI or transfected with DNM-PKC{epsilon} or vector alone for 48 h without serum, and cell lysates were analyzed by Western blotting using phospho-Ser-158-specific Lck antibody. Note the phosphorylation at 60 kDa only. (H) Activated Lck-Ser-158 in infected human Schwann cells directly phosphorylates Erk2. (Left; arrows) Lysates of control and M. leprae-infected Schwann cells were immunoprecipitated with phospho-Lck-Ser-158 antibody, phospho-PKC{alpha}/{beta}II antibody, or rabbit IgG, and precipitates were incubated with recombinant Erk2 in kinase buffer and then blotted with phospho-Erk1/2 antibody. (Right) The detection of phosphorylation of recombinant Erk2 by Lck-Ser-158 precipitated from Schwann cells using anti-phospho-Tyr antibody is shown. The same amount of recombinant Erk2 was incubated in kinase buffer without cell lysates and blotted with anti-phospho-tyrosine antibody. Note the Erk1/2 phosphorylation by activated Lck from infected Schwann cells.


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Fig. 5. Lck regulates M. leprae-induced nuclear accumulation of cyclin D1 and S-phase cells. (A) Thirty-day-infected human primary Schwann cells were serum-starved for 48 h in the continuous presence of MEKI and PI3KI with or without LckI, and immunoblots of the nuclear extracts were labeled with antibody to cyclin D1. Lamin A/C labeling is shown to indicate the purity of nuclear fraction and equal protein loading. (B) Cell cycle FACS analysis showing net M. leprae-induced S-phase cells (subtracted from synchronized control Schwann cells) in the continuous presence of MEKI and PI3KI with or without LckI. Data are presented from three independent experiments. *, P < 0.001, Student's t test. (C) A proposed model of Lck-mediated Erk1/2 activation and subsequent proliferation of human Schwann cells by intracellular M. leprae from inside the cells independent of the canonical Raf/MEK-dependent pathway.


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