Leptin receptor JAK2/STAT3 signaling modulates expression of Frizzled receptors in articular chondrocytes.
OBJECTIVE
Differentiated articular chondrocytes express a functional bisoform of the leptin receptor (LRb); however, leptin-LRb signaling in these cells is poorly understood. We hypothesized that leptin-LRb signaling in articular chondrocytes functions to modulate canonical Wnt signaling events by altering the expression of Frizzled (FZD) receptors.


METHODS
Human chondrocyte cell lines and primary articular chondrocytes were grown in serum containing growth media for 24h, followed by a media change to Dulbecco's modified Eagle's medium (DMEM) containing 1% Nutridoma-SP to obtain a serum-deficient environment for 24h before treatment. Treatments included recombinant human leptin (10-100nM), recombinant human IL-6 (0.3-3nM), or recombinant human erythropoietin (Epo) (10mU/ml). Cells were harvested 30min-48h after treatment and whole cell lysates were analyzed using immunoblots or luciferase assays.


RESULTS
Treatment of cells with leptin resulted in activation of Janus kinase 2 (JAK2) and subsequent phosphorylation of specific tyrosine residues on LRb, followed by dose- and time-dependent increases in the expression of Frizzled-1 (FZD1) and Frizzled-7 (FZD7). Leptin-mediated increases in the expression of FZD1 were blocked by pre-treatment with the protein synthesis inhibitor cycloheximide or the JAK2 inhibitor AG490. Experiments using a series of hybrid Epo extracellular domain-leptin intracellular domain receptors (ELR) harboring mutations of specific tyrosine residues in the cytoplasmic tail showed that increases in the expression of FZD1 were dependent on LRb-mediated phosphorylation of STAT3, but not ERK1/2 or STAT5. Leptin pre-treatment of chondrocytes prior to Wnt3a stimulation resulted in an increased magnitude of canonical Wnt signaling.


CONCLUSIONS
These experiments show that leptin-LRb signaling in articular chondrocytes modulates expression of canonical Wnt signaling receptors and suggests that direct cross-talk between these pathways is important in determining chondrocyte homeostasis.
Ohba
S
S
Division of Rheumatology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-5688, USA.
Lanigan
T M
TM
Roessler
B J
BJ
D000368:Aged; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D051157:Frizzled Receptors / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D053614:Janus Kinase 2 / Q000502:physiology*; D020738:Leptin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D050796:STAT3 Transcription Factor / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D051153:Wnt Proteins / Q000494:pharmacology / Q000502:physiology; D060508:Wnt3 Protein; D060509:Wnt3A Protein
D051157:Frizzled Receptors; D015850:Interleukin-6; D020738:Leptin; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C555510:WNT3A protein, human; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D048049:Extracellular Signal-Regulated MAP Kinases