Title

Evidence that P2 purinergic signaling in astrocytes regulates expression of N-cadherin and thrombospondin-1: Implications for CNS injury and development

Date of Award

2005

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Neuroscience

First Committee Member

Ellen Barrett, Committee Chair

Abstract

Purinergic regulation of N-cadherin and thrombospondin-1 (TSP-1), molecules produced by astrocytes that are involved in cell-cell and cell-matrix interactions, is unknown. I used primary cultures of rat cortical astrocytes to study the effects of purinergic signaling on the expression of N-cadherin and TSP-1. N-cadherin and TSP-1 protein expression increased in a time- and concentration-dependent manner with extracellular ATP. Cultures treated with a P1 antagonist, 8-(p-sulfophenyl)-theophylline, or P2 antagonists, reactive blue 2 or pyridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid (PPADS), showed reduced ATP-induced expression of N-cadherin and TSP-1, suggesting P1 and P2 receptors involvement in the regulation of N-cadherin and TSP-1 expression. However, agonists studies showed that UTP and 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), but not 2-methylthioADP (2MeSADP), adenosine, and N6-cyclopentyladenosine, statistically increased N-cadherin expression, while UTP but not BzATP, 2MeSADP, adenosine or N-ethylcarboxamidoadenosine statistically increased TSP-1 expression, suggesting that ATP-induced N-cadherin expression is mediated by both P2Y and P2X receptors while ATP-induced TSP-1 expression is mediated by P2Y receptors. Using various protein kinase pathway inhibitors, I found that the ATP-induced N-cadherin expression involves both extracellular signal-regulated protein kinase (ERK) and protein kinase B/Akt. However, other signaling pathways may also be involved since inhibition of both ERK and Akt did not completely block the ATP-induced N-cadherin expression. I found that ERK, Akt, p38-mitogen-activated protein kinase (MAPK) and c-Jun N-terminal protein kinase/stress activated-protein kinase were all involved in the ATP-induced TSP-1 expression, particularly p38/MAPK and Akt. Using an in vitro model of CNS mechanical injury, N-cadherin and TSP-1 expressions increased in a time-dependent manner after mechanical strain. Injury-induced TSP-1 expression involves P2 receptor activation since PPADS was able to block the injury-induced TSP-1 expression. In vivo studies with moderate traumatic brain injury showed that the expression of various P2 receptors changes with injury, particularly P2X 1 and P2X7 with increased mRNA but decreased protein levels. I have identified the signaling mechanisms involved in regulating N-cadherin and TSP-1 expression in rat astrocytes. These studies suggest that purinergic signaling may be an important factor in N-cadherin and TSP-1 mediated cell-matrix and cell-cell interactions such as occur during development and after CNS injury.

Keywords

Biology, Neuroscience

Link to Full Text

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