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Carbonic Anhydrase 2 is Required for Maintaining Metabolic Homeostasis of Human Articular Chondrocytes

ABSTRACT: Objective: To examine the role of carbonic anhydrase II (CA2 gene), the most efficient isoform in the CA family of zinc-containing metalloenzymes, in chondrocyte metabolism. Methods: CA2 expression was measured in human and mouse osteoarthritic cartilage. RNA-seq was performed in C28/I2 cells in which CA2 was silenced. Normal human chondrocytes were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen) following CA2 knockdown. Cell metabolism was studied by measuring extracellular lactate production, glucose consumption, intracellular ADP/ATP, intracellular pH, and ROS production. Glycolysis was measured using Seahorse XF96 analyzer. The effect of CA2 knockdown on anabolic and catabolic markers was measured following treatment with IL-1. Effects of CA2 was examined on cell proliferation, cell-cycle distribution, colony-formation, and migration. Results: CA2 was highly elevated in human and murine osteoarthritic cartilage. RNA-seq analysis revealed that processes related to glycolysis, apoptosis and TNF signaling were perturbed in cells lacking CA2. CA2 expression was 10-fold higher under hypoxia and its knockdown caused decreased extracellular lactate production, increased ADP/ATP ratio, impaired glycolysis, decreased glycolytic capacity and lowered the expression of glycolysis rate-limiting enzymes, but did not affect intracellular pH and ROS production. CA2 deficiency disturbed chondrocyte anabolic and catabolic equilibrium. CA2 knockdown suppressed chondrocyte migration and proliferation and induced cell-cycle arrest. Conclusion: This study unravels a novel role of CA2 in chondrocyte metabolism and inflammation. These findings indicate that CA2 is required for the maintenance of chondrocyte metabolic homeostasis. Future work is needed to further illuminate the mechanistic and functional role of CA2 in osteoarthritis development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE184062 | GEO | 2022/03/17

REPOSITORIES: GEO

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Project description:PAO1 was cultured planktonically to stationary phase with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.

Project description:PAO1 and FRD1 were cultured planktonically and in biofilms with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined for PAO1 cultured planktonically, for FRD1 cultured planktonically, for PAO1 cutured in biofilms, and for FRD1 cultured in biofilms. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.

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Carbonic Anhydrase 2 is Required for Maintaining Metabolic Homeostasis of Human Articular Chondrocytes

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