Compartmentalized phosphodiesterase 4D isoforms expression, targeting and localization in vascular myocytes
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During the development of atherosclerosis, contractile vascular smooth muscle cells (VSMCs) change to cells capable of migrating and proliferating to mediate repair, where the responses may be adaptive or mal-adaptive in effect. Cyclic adenosine monophosphate (cAMP)-elevating agents have been shown to inhibit migration of VSMC. cAMP activity within the cell is known to be ubiquitous and dynamic, requiring control through signal termination mechanisms for cellular homeostasis. Phosphodiesterase (PDE) enzymes are central to this critical regulatory process catalyzing the hydrolysis of cAMP. A great deal of insight into the role of PDEs in defining compartmentalization of cAMP signaling has arisen predominately from recent studies on the cAMP-specific PDE4 family. Compartmentalization of PDE4 is mediated by their unique N-terminal domains, which have been proposed to provide the “postcodes/zipcodes” for cellular localization. PDE4D isoforms vary widely, yet their conservation over evolutionary time suggests important non-redundant roles in distinct cellular processes. To study the potential role of individual PDE4D isoforms we seek to utilize the unique N-terminal targeting domains that are proposed to be responsible for their protein-protein interactions and site-directed localization. Herein, we report on the expression, targeting and localization of five “long” PDE4D isoforms and the impact on cell morphology of certain amino-terminal domains of individual PDE4D constructs expressing green fluorescent protein (NT-PDE4D/GFP) in human aortic smooth muscle cells (HASMCs). Through the development of engineered NT-PDE4D/GFP expression plasmids, we were able to study the cell biological impacts associated with the overexpression of individual PDE4D amino-terminal variants in HASMCs. We show that NT-PDE4D5/GFP and NT-PDE4D7/GFP expressing cells exhibited an elongated cell morphology, where this effect was much more marked in NT-PDE4D7/GFP expressing cells, exhibiting multiple leading edge structures and highly elongated “tails”. We identify a potential role for PDE4D7 targeting in the regulation of cell polarity and migration. Our results suggest the novel idea that PDE4D7, rather than the four other long PDE4D isoforms (PDE4D3, PDE4D5, PDE4D8, or PDE4D9), represents the dominant PDE4D variant involved in controlling cAMP-mediated effects on cell tail retraction dynamics.