(381) Carbonic Anhydrase12 Knockout Mimics CF Cellular Phenotypes

Background: Carbonic anhydrases (CA) reversibly produce bicarbonate and a proton from carbon dioxide and water. There are up to 15 CAs that have been identified and they play a critical role in maintaining cellular bicarbonate levels and pH. The key evidence linking CA function to CF phenotypes is from studies demonstrating that patients with mutations in CA12 exhibit CF-like disease including elevated sweat chloride levels despite perfectly functioning CFTR (1). In particular, an adult proband exhibited lung disease with multiple pulmonary exacerbations, Pseudomonas-positive sputum cultures, and mild bronchiectasis. There are multiple mechanistic possibilities that could link CA12 to CF-like phenotypes such as extracellular pH regulation. In this study it is hypothesized that CA12 deficiency will mimic cellular CF phenotypes such as intracellular transport. We have recently shown that CA2 deficiency in CF cells contributes to these CF phenotypes supporting the role of bicarbonate regulation as a key modulator of CF cellular function (2).

Methods: In this study, we created a CA 12 knockout cell line (KO) and its control cell line (Ctrl) based on 9HTEo- human tracheal epithelial cell line using CRISPR-Cas9 technique. Intracellular transport was monitored by filipin staining to follow cholesterol trafficking and rab7 immunostaining to directly monitor endosomal movement.

Results: Our previous studies have demonstrated that CF cells exhibit increased perinuclear cholesterol accumulation, decreased microtubule (MT) reformation rate, and limited endosomal distribution. We examined the above phenotypes of KO and Ctrl cells. Over 100 cells were quantified for each group in each experiment method. We first examined cholesterol distribution via filipin staining. The percentage of cells with cholesterol accumulation was much higher in KO cells (73.6 ± 7.3%) when compared to Ctrl (21.4 ± 5.9%, p < 0.001). Next, we examined the MT reformation by depolymerizing MTs in the cells and observing the reformation of MTs at different time points. At 2 min and 4 min, the percentages of cells with MT asters were 17.4 ± 5.3% and 49.0 ± 16.9% for KO and 38.1 ± 8% and 66.3 ± 11.9% for Ctrl. The MT reformation of KO was significantly slower than that of Ctrl at 2 min (p < 0.001) and 4 min (p < 0.05). Then, we examined the endosomal distribution in these cells by Rab7 immunocytochemistry. The mean percentage of Rab7 covered nuclear circumferences was 53.2% for KO and 89.6% for Ctrl. All these data demonstrated that the characteristics of Ctrl were similar to WT 9HTEo-, and KO showed CF-like properties. Last we tested treatment efficacies of 3 CF-effective drugs 8-CPT-2Me-cAMP, AICAR, and tubastatin. After 24 hours of treatment, all of the 3 drugs slightly decreased the cholesterol accumulation and increased the endosomal distribution of KO cells, though still significantly different from Ctrl cells, which suggested these drugs improved the intracellular transport in KO but could not fully recover the damage caused by CA12 KO.

Conclusions:

Conclusions: These findings show that depletion of CA12 expression mimicks CF cellular phenotypes without impacting CFTR function. These data point to mechanisms key to CF cellular phenotypes and could explain some of the CF clinical similarities in patients with CA12 mutations.


Acknowledgements: Supported by CFF.

References: 1. 1. Lee M, et al. (2016) Hum Mol Gen, 1923-33.

2. 2. Boyne K, et al. (2022) AJP Lung, L33-L347.