טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLahad Iris
SubjectInvolvement of MAPK Signal Transduction
Pathways in Human Monocyte Differentiation
DepartmentDepartment of Medicine
Supervisor Professor Shimon Pollack


Abstract

Mitogen-activated protein kinase (MAPK) signal transduction pathways are involved in human monocyte differentiation. However, the precise mode of involvement and the interactions between the 3 different MAPKs (ERK, JNK, p38) is scarcely known.  We have investigated the differential involvement of ERK, JNK and p38 in PMA-induced differentiation of human THP-1 monocytes (PMA-MAC) and in myelomonocytic cell lines (HL-60 and U937).  MAPKs activity was assayed by western immunoblotting, phosphotransferase assay and in-vitro kinase assay. The role of each MAPK signal transduction pathway in the differentiation process was evaluated using specific MAPK inhibitors. Gene Array technology was applied to analyze gene transcripts (mRNA) of MAPK enzymes and cytokines. Secretion of cytokines from PMA-MAC was measured by ELISA.  Variable ERK activation patterns were observed in phorbol-ester (PE)-stimulated human monocytic cell lines suggesting that different monocyte maturation states may be associated with unique ERK activation patterns. Addition of specific PKC or MEK inhibitors to PE-stimulated monocytes was associated with a significant decrease in MAPK activity, which may suggest the use of the PKC-MEK-ERK pathway in PE-induced human monocytes activation. Differentiation of human monocytes to PMA-MAC is associated with the involvement of ERK1/2 (especially ERK2) since the early stages of differentiation whereas JNK (especially JNK2) phosphorylation is enhanced towards later stages of differentiation. p38 is not involved in the differentiation process.  The addition of MEK inhibitor to differentiating THP-1 cells was associated with enhancement of p38 phosphorylation and vice-versa. This may suggest a cross-talk between the MAPK pathways in THP-1 differentiation. Differentiated THP-1 cells produce and secret different pro-inflammatory cytokines. ERK inhibition resulted in an almost complete abrogation of cytokine secretion except TGF-b. JNK inhibition resulted in significantly reduced secretion of all cytokines except IL-12, IL-18 and TGF-b, whereas p38 inhibition does not suppress any cytokine, except IL-6. We conclude that PMA-induced human monocyte differentiation is accompanied by ERK and JNK activation in different stages of the maturation process. p38 does not participate directly in the differentiation process but may regulate ERK pathway. The secretion of adaptive pro-inflammatory cytokines (TNFa, IL-1b, IL-8, IL-6) from PMA-MAC is mediated by both ERK and JNK pathways whereas the secretion of innate pro-inflammatory cytokines (IL-12, IL-18) is mediated only by ERK pathway. TGF-b secretion is not mediated by any of the MAPK pathways.