CAMKK2 is upregulated in primary human osteoarthritis and its inhibition protects against chondrocyte apoptosis

J E Dilley; A Seetharam; X Ding; M A Bello; J Shutter; D B Burr; R M Natoli; T O McKinley; U Sankar

doi:10.1016/j.joca.2023.02.072

CAMKK2 is upregulated in primary human osteoarthritis and its inhibition protects against chondrocyte apoptosis

Osteoarthritis Cartilage. 2023 Jul;31(7):908-918. doi: 10.1016/j.joca.2023.02.072. Epub 2023 Feb 27.

Authors

J E Dilley¹, A Seetharam², X Ding³, M A Bello⁴, J Shutter⁵, D B Burr⁶, R M Natoli⁷, T O McKinley⁸, U Sankar⁹

Affiliations

¹ Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: jedilley@indiana.edu.
² Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: abhiseet@iu.edu.
³ Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: xinding@iu.edu.
⁴ Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: belloma@iu.edu.
⁵ Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: jeshut@iu.edu.
⁶ Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: dburr@iu.edu.
⁷ Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: rnatoli@iuhealth.org.
⁸ Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: tmckinley@iuhealth.org.
⁹ Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: usankar@iu.edu.

PMID: 36858195
PMCID: PMC10272098 (available on 2024-07-01)
DOI: 10.1016/j.joca.2023.02.072

Abstract

Objective: To investigate the role of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) in human osteoarthritis.

Materials and methods: Paired osteochondral plugs and articular chondrocytes were isolated from the relatively healthier (intact) and damaged portions of human femoral heads collected from patients undergoing total hip arthroplasty for primary osteoarthritis (OA). Cartilage from femoral plugs were either flash frozen for gene expression analysis or histology and immunohistochemistry. Chondrocyte apoptosis in the presence or absence of CAMKK2 inhibition was measured using flow cytometry. CAMKK2 overexpression and knockdown in articular chondrocytes were achieved via Lentivirus- and siRNA-mediated approaches respectively, and their effect on pro-apoptotic and cartilage catabolic mechanisms was assessed by immunoblotting.

Results: CAMKK2 mRNA and protein levels were elevated in articular chondrocytes from human OA cartilage compared to paired healthier intact samples. This increase was associated with elevated catabolic marker matrix metalloproteinase 13 (MMP-13), and diminished anabolic markers aggrecan (ACAN) and type II collagen (COL2A1) levels. OA chondrocytes displayed enhanced apoptosis, which was suppressed following pharmacological inhibition of CAMKK2. Levels of MMP13, pSTAT3, and the pro-apoptotic marker BAX became elevated when CAMKK2, but not its kinase-defective mutant was overexpressed, whereas knockdown of the kinase decreased the levels of these proteins.

Conclusions: CAMKK2 is upregulated in human OA cartilage and is associated with elevated levels of pro-apoptotic and catabolic proteins. Inhibition or knockdown of CAMKK2 led to decreased chondrocyte apoptosis and catabolic protein levels, whereas its overexpression elevated them. CAMKK2 may be a therapeutic target to prevent or mitigate human OA.

Keywords: Apoptosis; CAMKK2; Catabolism; Chondrocyte; Osteoarthritis.

MeSH terms

Apoptosis
Calcium-Calmodulin-Dependent Protein Kinase Kinase / genetics
Cartilage, Articular* / pathology
Cells, Cultured
Chondrocytes / metabolism
Humans
Osteoarthritis* / metabolism

Substances

CAMKK2 protein, human
Calcium-Calmodulin-Dependent Protein Kinase Kinase

Abstract

MeSH terms

Substances

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