sentence: Treatments included recombinant human leptin ( 10 - 100nM ) , recombinant human IL-6 ( 0.3 - 3nM ) , or recombinant human erythropoietin ( Epo ) ( 10mU/ml ) .
paragraph: Leptin receptor JAK2/STAT3 signaling modulates expression of Frizzled receptors in articular chondrocytes. 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. ### conclusion 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.