ALBERT

Description: Physiological mechanical loading is crucial for maintenance of bone integrity and architecture. We have calculated the strain caused by gravity stress on osteoblasts and found that 4-30g corresponds to physiological levels of 40-300 microstrain. Short-term gravity loading (15 minutes) induced a 15-fold increase in expression of growth-related immediate early gene c-fos, a 5-fold increase in egr-1, and a 3-fold increase in autocrine bFGF. The non-growth-related genes EP-1, TGF-beta, and 18s were unaffected by gravity loading. Short-term physiological loading induced extracellular signal-regulated kinase (ERK 1/2) phosphorylation in a dose-dependent manner with maximum phosphorylation saturating at mechanical loading levels of 12g (p 〈 0.001) with no effect on total ERK. The phosphorylation of focal adhesion kinase (FAK) was unaffected by mechanical force. g-Loading did not activate P38 MAPK or c-jun N-terminal kinase (JNK). Additionally, a gravity pulse resulted in the localization of phosphorylated ERK 1/2 to the nucleus; this did not occur in unloaded cells. The induction of c-fos was inhibited 74% by the MEK1/2 inhibitor U0126 (p 〈 0.001) but was not affected by MEK1 or p38 MAPK-specific inhibitors. The long-term consequence of a single 15-minute gravity pulse was a 64% increase in cell growth (p 〈 0.001). U0126 significantly inhibited gravity-induced growth by 50% (p 〈 0.001). These studies suggest that short periods of physiological mechanical stress induce immediate early gene expression and growth in MC3T3-E1 osteoblasts primarily through an ERK 1/2-mediated pathway.

Description: Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p〈0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and MAPK pathways plays a role in early T-cell activation and induction of IL-2, IL-2R(alpha) and IFN(gamma) gene expression.