Abstract

Cyclic nucleotide signaling pathway plays a significant role in various biological processes such as cell growth, transcription, inflammation, in microbial pathogenesis, etc. Modulation of cyclic nucleotide levels by optogenetic tools has overcome certain limitations of studying transduction cascade by pharmacological agents and has allowed several ways to modulate biological processes in a spatiotemporal manner. Here, we have shown the optogenetic modulation of the cyclooxygenase 2 (Cox-2) gene expression and their downstream effector molecule (PGE2) in HEK-293T cells and the development process of Dictyostelium discoideum via modulating the cyclic nucleotide (cAMP) signaling pathway utilizing photoactivated adenylyl cyclases (PACs) as an optogenetic tool. Light-induced activation of PACs in HEK-293T cells increases the cAMP level that leads to activation of cAMP response element-binding protein (CREB) transcription factor and further upregulates downstream Cox-2 gene expression and their downstream effector molecule prostaglandin E2. In D. discoideum, the light-regulated increase in cAMP level affects the starvation-induced developmental process. These PACs could modulate the cAMP levels in a light-dependent manner and have a potential to control gene expression and their downstream effector molecules with varying magnitude. It would enable one to utilize PAC as a tool to decipher cyclic nucleotide mediated signaling pathway regulations and their mechanism.

Highlights

  • Cells respond to the external stimuli using complex molecular signaling cascades for transmitting information from the external environment into the cell

  • We showed that the different photoactivated adenylyl cyclases (PACs) exhibited light regulated adenylyl cyclase activity, which in turn could control the activation of the cyclic AMP (cAMP) response element binding protein (CREB) transcription factor leading to the expression of the downstream gene (Cox-2) and their associated downstream effector molecule (PGE2) in HEK-293T cells

  • For expression in human cell lines, codon optimized synthetic genes, NgPAC1, NgPAC2, NgPAC3, and bPAC, were cloned into the pA3M mammalian expression vector and expressed in HEK-293T cells as a C-terminally tagged myc tag fusion protein (PAC::myc)

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Summary

Introduction

Cells respond to the external stimuli using complex molecular signaling cascades for transmitting information from the external environment into the cell. The regulatory effects of cAMP in eukaryotes are mediated via various downstream effector proteins such as Protein kinase A (PKA)[6], Exchange Protein directly Activated by cAMP (EPAC)[7], Cyclic Nucleotide-gated ion channels (CNG)[8] These effector molecules sense the changes in intracellular cAMP levels and regulate numerous cellular responses[9,10,11,12]. We employed the PACs to manipulate the cAMP-signaling pathway and analyzed the cAMP-mediated CREB-dependent gene expression in response to blue light. We showed that the different PACs exhibited light regulated adenylyl cyclase activity, which in turn could control the activation of the CREB transcription factor leading to the expression of the downstream gene (Cox-2) and their associated downstream effector molecule (PGE2) in HEK-293T cells. We observed that starvation-induced development was affected in cells expressing PACs, as a result of alteration in the cAMP level in response to light

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