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The cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family plays a major role in the expression of skeletal-specific genes and skeletal tissue development. We analyzed the changes of the amount, degree of phosphorylation and binding activity of the CREB/ATF family in the course of development of the murine calvarial osteoblastic cell line MC3T3-E1 as an in vitro model system of bone formation. The amount of CREB in the whole-cell extract detectable by Western blot analysis was high through all stages of development and maximal in the proliferation stage. The degree of phosphorylation estimated with anti-phosphorylated CREB antibody changed greatly and reached high levels in the proliferation stage and early mineralization stage. The ratio of phosphorylated CREB to total CREB in the CREB-CRE complex was also examined by gel shift assay. Although the binding to the consensus/CRE probe reached almost equally high levels in the proliferation stage and early mineralization stage, the relative level of phosphorylated CREB in the CREB-CRE complex was different in these two stages. In the early mineralization stage, most CREB bound to consensus/CRE was phosphorylated, while both phosphorylated and unphosphorylated CREB were bound to consensus/CRE in the proliferation stage. ATF-1 was also detected as a minor component bound to the consensus/CRE probe. The alteration of the binding of CREB to consensus/CRE over the course of osteoblast development supports the hypothesis that CREB may regulate the expression of genes defining the developmental sequence of MC3T3-E1 cells.

High mobility group box-1 protein (HMGB-1), a protein expressed highly in developing neurons, is involved in the development and differentiation of neurons. At the same time, it functions as a transcriptional regulator of particular genes and as a cytokine: HMGB-1 released from a defective cell has been reported to induce damage to the adjacent cells.With a view to examine the relationship between neuronal damage caused by hydrocephalus and HMGB-1, we analyzed the expression of HMGB-1 in the cerebellum, cerebrum, and hippocampus of 1-day-old congenitally hydrocephalic H-Tx rats.As opposed to nonhydrocephalic H-Tx rats, the hydrocephalic H-Tx rats were observed to show stronger expression of HMGB-1 in the cerebellum, cerebrum, and hippocampus. Consequently, the protein was presumed to influence the development of neurons from an early postnatal stage not only in the cerebral cortex and hippocampus but also in the cerebellum, which is less susceptible to the direct effects of hydrocephalus. We expect that, in the future, regulating the expression or functions of HMGB-1 will lead to the possibility of impeding the progress of neuronal damage caused by hydrocephalus.

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