A Relationship between p27kip1and Skp2 after Adult Brain Injury: Implications for Glial Proliferation

Abstract

S-phase-associated kinase protein-2 (Skp2) is involved in ubiquitination and proteasome-mediated degradation of p27(kip1), which plays an important role in mammalian cell-cycle regulation and neurogenesis in the developing central nervous system. To investigate their expression and function in central nervous system injury and repair, we used a brain-penetrating injury model in adult rats. Western blot analysis showed a significant downregulation of p27(kip1) and a concomitant upregulation of Skp2 following brain injury, and their expression profiles were temporally correlative (r = -0.910, p = 0.037). Immunofluorescence double-labeling revealed that p27(kip1) was highly expressed in neurons (51%), astrocytes (72%), and microglia (76%) in the sham group, while its expression was decreased prominently in microglia (26%) and astrocytes (32%) at 3 days after injury. Meanwhile, Skp2 expression was very low in all cell types in the sham group; however, 3 days after injury, its expression was increased significantly in microglia (51%) and astrocytes (31%) (p < 0.001), and less significantly in neurons (8%) (p = 0.038), and the astrocytes and microglia had proliferated. We also examined the expression profiles of CDK2, threonine-187 phosphorylated p27(kip1), proliferating cell nuclear antigen (PCNA), and Ki67, and their changes correlated with the expression profiles of p27(kip1) and Skp2. Moreover, co-immunoprecipitation data suggested that the protein-protein interactions between p27(kip1) and Skp2 were enhanced after injury. Taken with results of previous reports, we hypothesize the Skp2 is related to the downregulation of p27(kip1) expression after brain injury, and such an event may be associated with glial proliferation, including that of astrocytes and microglia.