| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
We, previously, reported that the transferrin receptor (Trf-R) positive (Trf-R+) and negative (Trf-R-) cells appeared after treatment with dimethyl sulfoxide (Me2SO) or retinoic acid, and commitment to neutrophilic differentiation and proliferation in Me_2SO-treated HL-60 cells. To clarify the molecular mechanism of commitment to differentiation and proliferation in both-type cells, we compared the protein expression profiles in Trf-R+ and Trf-R- cells using two-dimensional polyacrylamide electrophoresis. While 55kDa and 25kDa spots were more abundant in Trf-R+ cells, the expression of 11kDa spot is apparently increased in Trf-R- cells than that in Trf-R+ cells. The MALDI-MS spectrum analysis and Edman analysis revealed that the 11kDa protein is Calgranulin/S100A8 protein. Knock-down of S100A8 protein using siRNA inhibited the differentiation of HL-60 cells induced by Me_2SO, suggesting that S100A8.
Since atypical PKC(aPKC) has been reported to play an important role on cell polarity, w
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e focused to clarify the role of aPKC on differentiation and proliferation of neutrophilic maturation. Particularly, we analyzed the participation of aPKC, PKCι/λ in relation to G-CSF-dependent proliferation and S6K1 and PI3K activation. Protein kinase Cι was abundantly expressed in HL-60 cells, but not PKCζ ; the maximum stimulation of PKCι, was observed from 15 min to 30 min after the addition of G-CSF. An immuno-precipitation assay using an anti-PKCι antibody revealed that PKCι formed a complex with S6K1 30 min after the addition of G-CSF. There was a time lag between the activation of PI3K and S6K1 when G-CSF was added to the HL-60 cells. Within 5 to 15 min during this lag time, PKCι was found to translocate from the nucleus to the membrane, and re-translocate to the cytosol, resulting in the association with S6K1. Small interfering RNA for PKCι inhibited G-CSF-induced proliferation and phosphorylation of Thr-389 of S6K1,and promoted differentiation. The dynamic translocation and activation processes of PKCι can be explained as the reason for the time lag in maximum activation between PI3K and S6K1.
These results indicate that while S100A8 plays an important role on differentiation of HL-60 cells., PI3K/PKCι/p70S6K cascade contribute main role on the proliferation of differentiating HL-60 cells. Less
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