Carbonic anhydrase and soluble adenylate cyclase regulation of cystic fibrosis cellular phenotypes.

Kathleen Boyne,Binyu Lu,Thomas J Kelley,Walter F Boron,Deborah A Corey,Pan Zhao,Fraser J Moss

doi:10.1152/ajplung.00022.2021

Abstract

Several aspects of the cell biology of cystic fibrosis (CF) epithelial cells are altered including impaired lipid regulation, disrupted intracellular transport, and impaired microtubule regulation. It is unclear how the loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to these differences. It is hypothesized that the loss of CFTR function leads to altered regulation of carbonic anhydrase (CA) activity resulting in cellular phenotypic changes. In this study, it is demonstrated that CA2 protein expression is reduced in CF model cells, primary mouse nasal epithelial (MNE) cells, excised MNE tissue, and primary human nasal epithelial cells (P < 0.05). This corresponds to a decrease in CA2 RNA expression measured by qPCR as well as an overall reduction in CA activity in primary CF MNEs. The addition of CFTR-inhibitor-172 to WT MNE cells for ≥24 h mimics the significantly lower protein expression of CA2 in CF cells. Treatment of CF cells with l-phenylalanine (L-Phe), an activator of CA activity, restores endosomal transport through an effect on microtubule regulation in a manner dependent on soluble adenylate cyclase (sAC). This effect can be blocked with the CA2-selective inhibitor dorzolamide. These data suggest that the loss of CFTR function leads to the decreased expression of CA2 resulting in the downstream cell signaling alterations observed in CF.

Highlights

Cystic Fibrosis (CF) is a progressive, autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene
To test whether the effects of KH7 were mediated through EPAC1 as we have shown in CF cells, we treated cells with the EPAC1 selective activator 8-cpt-2-O-Me-cAMP (8-cpt-cA) to see if that would overcome the effects of soluble adenylate cyclase (sAC) inhibition
In this study, we have shown that sAC is a key regulator of processes that are altered in CF cells including microtubule dynamics and intracellular transport

Summary

Introduction

Cystic Fibrosis (CF) is a progressive, autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Reduced or dysfunctional CFTR leads to decreased Cl– transport and increased Na+ transport across epithelium, and to alterations in several aspects of the cell biology of CF epithelial cells. We have recently identified that microtubule regulation is altered in CF cells consisting of reduced tubulin acetylation and slower rates of reformation [27, 29]. These alterations result in disrupted intracellular transport marked by reduced endosomal movement and accumulation in the perinuclear region [45]. Microtubule dysfunction and intracellular transport can be restored in CF cells by inhibiting histone deacetylase 6 (HDAC6), a cytosolic deacetylase that regulates tubulin acetylation among other targets [32]. Since evidence suggests that microtubule regulation is key to important CF phenotypes, we need to understand in more detail how the absence of CFTR function leads to microtubule changes

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Results

Conclusion