Amal I. Malik, Ph.D. Biology, 2009

Cellular adaptation to dehydration stress: Molecular adaptations for dehydration tolerance in the African clawed frog, Xenopus laevis

 

Abstract:

The thesis addressed multiple questions about the signal transduction mechanisms that trigger gene expression responses to dehydration signals, and about the role of antioxidant defenses in combating dehydration stress in the African clawed frog, Xenopus laevis. In the first part of the thesis the responses to dehydration stress by the three main MAPK cascades were traced by measuring both total protein levels, and the relative amounts of active phosphorylated proteins for multiple intermediates in the p38 MAPK, stress-activated protein kinase (SAPK), and extracellular signal-regulated kinase (ERK) cascades. The data documented a major activation of the ERK pathway in most organs of X. laevis during dehydration. Selected upstream activator and downstream targets of the ERK pathway also showed pronounced tissue specific regulation in response to dehydration. The SAPK was activated in skeletal muscle, lung, and skin whereas the p38 MAPK was activated in the lung and kidney of X. laevis.  The second part of the thesis focused on antioxidant defenses that are known to be contributors to cell preservation under various types of stress. Two main transcription factors that regulate antioxidant genes were activated in response to dehydration in X. laevis organs:  NF-E2 related factor (Nrf2) and forkhead box, class O (FoxO). Immunoblottingshowed a significant increase in their nuclear translocation, and enzyme-linked immunosorbant assays showed increased DNA binding activity by FoxO1 under dehydration stress. Expression of downstream target genes controlled by these transcription factors was enhanced during dehydration. Six family members of the glutathione S-transferase (GST) and three family members of the aldo-keto reductase (AKR) showed tissue specific expression, correlated with Nrf2 activation. Manganese-dependent superoxide dismutase (MnSOD) and catalase expression were also elevated under FoxO1 control. Improved antioxidant defenses may be critical to dealing with variations in tissue oxygenation and reactive oxygen species generation that are one consequence of large changes in body hydration that affect oxygen delivery to tissues. This thesis showed for the first time that the MAPKs family members are selectively activated in response to two levels of dehydration stress in X. laevis. Also, antioxidant defenses play a critical protective role during dehydration stress in these frogs.