C - Michigan Technological University

eyond the G1/S checkpoint, thus inducing proliferation of osteoblast progenitors (forreview see: [286, 287]). Expression of Cyclin D1 was expected to decrease inosteoblasts cultured in the serum from hibernating bears. Clock genes (e.g. Per1, Per2,Bmal1) control cell cycle progression past the G1/S and G2/M checkpoints. A halt in theG1 phase could be indirectly caused by an increase in Bmal1 through modulation ofc-myc and Cyclin D1 [285, 288, 289]. Alternatively, a halt in the G2 phase could be dueto an increase in Per1 [290]. Since a halt in either cell phase could slow proliferation andprevent excessive use of metabolic energy, an increase in either Per1 or Bmal1 inosteoblasts cultured in hibernating bear serum was expected.In the second hypothesis, the caspase-3/7 response of osteoblasts cultured inseasonal bear serum (observed in Chapter 5) was expected to be correlated to geneexpression of apoptosis regulators. In the Bcl-2 family, expression of anti-apoptotic Bcl-2and Bcl-XL were expected to increase in osteoblasts cultured in the serum of hibernatingbears, with a concurrent decrease in expression of pro-apoptotic BAK, BAX, and BAD.Likewise, the pro-survival gene Akt was expected to increase in osteoblast cultured inserum from hibernating bears. Thus, osteoblasts cultured in serum from hibernatingbears were expected to favor anti-apoptotic gene expression compared to cells culturedin serum from active bears. If such a decrease in apoptotic signaling occurred inosteoblasts of the hibernating bear, bone resorption could be suppressed during thisperiod of disuse.The third hypothesis of this study was that expression of osteoblast differentiationmarkers would decrease in cells cultured in hibernating serum (compared to cells inactive serum). The transcription factor Runx2 is upregulated during the differentiation ofosteoblasts (for review see: [291]), and was expected to be lower in osteoblasts culturedin serum from hibernating bears. Ubiquitin is a small regulatory protein which iscovalently bound to other proteins as a tag for proteolysis. It is often used as a referencegene because its expression is ubiquitous in all cell types. In osteoblasts, Smad ubiquitinregulatory factor 1 (Smurf1) ligates ubiquitin to Runx2 and other osteogenic regulators(for review see: [292]). The subsequent proteolysis of these tagged proteins suppressesosteoblast activity (i.e. matrix production, BSALP activity) [293, 294]. Smurf1 wasexpected to increase in osteoblasts cultured in hibernating bear serum thus reducing91