Dopamine suppresses osteoclast differentiation via cAMP/PKA/CREB pathway

How the nervous system regulates bone remodeling is an exciting area of emerging research in bone biology. Accumulating evidence suggest that neurotransmitter-mediated inputs from neurons may act directly on osteoclasts. Dopamine is a neurotransmitter that can be released by hypothalamic neurons to regulate bone metabolism through the hypothalamic-pituitary-gonadal axis. Dopamine is also present in sympathetic nerves that penetrate skeletal structures throughout the body. It has been shown that dopamine suppresses osteoclast differentiation via a D2-like receptors (D2R)-dependent manner, but the intracellular secondary signaling pathway has not been elucidated. In this study, we found that cAMP-response element binding protein (CREB) activity responds to dopamine treatment during osteoclastogenesis. Considering the critical role of CREB in osteoclastogenesis, we hypothesize that CREB may be a critical target in dopamine's regulation of osteoclast differentiation. We confirmed that D2R is also present in RAW cells and activated by dopamine. Binding of dopamine to D2R inhibits the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway which ultimately decreases CREB phosphorylation during osteoclastogenesis. This was also associated with diminished expression of osteoclast markers that are downstream of CREB. Pharmacological activation of adenylate cyclase (to increase cAMP production) and PKA reverses the effect of dopamine on CREB activity and osteoclastogenesis. Therefore, we have identified D2R/cAMP/PKA/CREB as a candidate pathway that mediates dopamine's inhibition of osteoclast differentiation. These findings will contribute to our understanding of how the nervous and skeletal systems interact to regulate bone remodeling. This will enable future work toward elucidating the role of the nervous system in bone development, repair, aging, and degenerative disease.     

膜去极化与cAMP在胰岛细胞株HIT中诱导CBP片段的转录活性

为了研究 Ｃ Ｂ Ｐ在胰岛 Ｈ Ｉ Ｔ 细胞中调节基因转录的机制,将不同的 Ｃ Ｂ Ｐ片段瞬时转染到细胞中,观察其转录活性．实验表明,在胰岛 Ｈ Ｉ Ｔ 细胞中,膜去极化及 ｃ Ａ Ｍ Ｐ 均可诱导 Ｃ Ｂ Ｐ３０（ Ｃ Ｒ Ｅ Ｂ结合功能区）转录活性增强,并有协同效应． Ｐ Ｋ Ｃ对 Ｃ Ｂ Ｐ３０ 的转录活性无影响;与 Ｃ Ｒ Ｅ Ｂ有更强结合力的 Ｃ Ｂ Ｐ Ｋ Ｉ Ｘ Ｓ／ Ｂ（氨基酸序列短于 Ｃ Ｂ Ｐ３０ 的 Ｃ Ｒ Ｅ Ｂ结合功能区）其基本转录活性及膜去极化、ｃ Ａ Ｍ Ｐ诱导下的转录活性均比 Ｃ Ｂ Ｐ３０ 更强．反义 Ｃ Ｒ Ｅ Ｂ 的过度表达可降低 ｃ Ａ Ｍ Ｐ诱导的 Ｃ Ｂ Ｐ的转录活性．提示在胰岛 Ｈ Ｉ Ｔ 细胞中,膜去极化及 ｃ Ａ Ｍ Ｐ对共转录因子 Ｃ Ｂ Ｐ转录活性的调节作用通过 Ｃ Ｒ Ｅ Ｂ介导．     

cAMP-induced cytoskeleton rearrangement increases calcium transients through the enhancement of capacitative calcium entry

In this report we investigated the correlation between cell morphology and regulation of cytosolic calcium homeostasis. Type I astrocytes were differentiated to stellate process-bearing cells by a 100-min exposure to cAMP. Differentiation of cortical astrocytes increased the magnitude and duration of calcium transients elicited by phospholipase C-activating agents as measured by single cell Fura-2-based imaging. Calcium imaging showed differences in the spatial pattern of the response. In both differentiated and the control cells, the response originated in the periphery and gradually extended into the center of the cell. However, the elevation of cytosolic calcium concentration ([Ca(2+)](i)) was particularly evident within the processes and adjacent to the inner cell membrane of the differentiated astrocytes. In addition, differentiation significantly prolonged the duration of the [Ca(2+)](i) elevation. Potentiation of the calcium transients was mimicked by forskolin-induced differentiation and abolished by a specific protein kinase-A blocker. Conversely, the enhancement of the calcium transients was not mimicked by brief exposure to cAMP not causing morphological differentiation, and in PC12 cells that did not undergo morphological changes after 100 min of cAMP treatment. Impairing cAMP-induced cytoskeleton re-organization, by means of cytochalasin D and nocodazole, prevented the potentiation of the calcium transients in cAMP-treated astrocytes. Phospholipase C activity and sensitivity to inositol (1,4,5)-trisphosphate were not involved in the enhancement of the calcium responses. Also, potentiation of the calcium transients was dependent on extracellular calcium. Calcium storage and thapsigargin-depletable intracellular calcium reservoirs were analogously not increased in differentiated astrocytes. Rearrangement of the cell shape also caused a condensation of the endoplasmic reticulum and altered the spatial relationship between the endoplasmic reticulum and the cell membrane. In conclusion, morphological rearrangements of type I astrocytes increase the magnitude and the duration of agonist-induced calcium transients via enhancement of capacitative calcium entry and is associated with a spatial reorganization of the relationship between cell membrane and the endoplasmic reticulum structures.     

Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.