Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells.

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. We reported previously that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling (Freedman et al., 2015). We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic, biochemical, and quantitative phosphopeptide analyses, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via a phosphorylated tyrosine (Y32) in its unique region. This distinct mode of c-Cbl recognition depresses steady-state expression of LynA in macrophages derived from mice. Mast cells, however, express little c-Cbl and have correspondingly high LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression thus builds cell specificity into the LynA checkpoint.

Biomarkers of inflammation in infants with cystic fibrosis.

BACKGROUND: There are urgent needs for clinically relevant biomarkers to identify children with cystic fibrosis (CF) at risk for more progressive lung disease and to serve as outcome measures for clinical trials. Our objective was to investigate three targeted biomarkers in a population of asymptomatic CF infants. METHODS: Urine, blood and lung function data were collected for 2 years from clinically stable infants diagnosed with CF by newborn screening. A subset of CF infants had bronchoscopy with lavage performed at 6 months and 1 year. Urine was collected quarterly from healthy control infants. Expectorated sputum and urine were collected quarterly for 2 years from clinically stable CF adults. Desmosine, club cell secretory protein (CCSP) and cathepsin B concentrations were measured and compared. Mixed effects models were used to identify associations between biomarker concentrations and clinical characteristics. Receiver operator characteristic curves were generated to investigate the sensitivity and specificity of the biomarkers. RESULTS: Urinary cathepsin B was significantly higher in CF infants compared to healthy infants (p = 0.005). CF infant airway and urinary cathepsin B concentrations were significantly lower compared to adult CF subjects (p = 0.002 & p = 0.022, respectively). CF infant airway CCSP was significantly higher than adult CF subjects (p < 0.001). There was a significant correlation between CF infant plasma CCSP and BALF CCSP (p = 0.046). BALF CCSP was negatively associated with IL-8 (p = 0.017). There was no correlation between biomarker concentration and FEV0.5. CONCLUSIONS: Cathepsin B and CCSP show promise as biomarkers of inflammation in CF infants. Further study is needed.

Modulation of musculoskeletal hyperalgesia by brown adipose tissue activity in mice.

Cold exposure and a variety of types of mild stress increase pain in patients with painful disorders such as fibromyalgia syndrome. Acutely, stress induces thermogenesis by increasing sympathetic activation of beta-3 (ß3) adrenergic receptors in brown adipose tissue. Chronic stress leads to the hypertrophy of brown adipose, a phenomenon termed adaptive thermogenesis. Based on the innervation of skeletal muscle by collaterals of nerves projecting to brown adipose, we theorized an association between brown adipose tissue activity and musculoskeletal hyperalgesia and tested this hypothesis in mice. Exposure to a cold swim or injection of BRL37344 (ß3 adrenergic agonist) each enhanced musculoskeletal hyperalgesia, as indicated by morphine-sensitive decreases in grip force responses, whereas SR59230A (ß3 adrenergic antagonist) attenuated swim-induced hyperalgesia. Chemical ablation of interscapular brown adipose, using Rose Bengal, attenuated the development of hyperalgesia in response to either swim stress or BRL37344. In addition, elimination of the gene expressing uncoupling protein-1 (UCP1), the enzyme responsible for thermogenesis, prevented musculoskeletal hyperalgesia in response to either a swim or BRL37344, as documented in UCP1-knockout (UCP1-KO) mice compared with wild-type controls. Together, these data provide a convergence of evidence suggesting that activation of brown adipose contributes to stress-induced musculoskeletal hyperalgesia.

Intrathecal urocortin I in the spinal cord as a murine model of stress hormone-induced musculoskeletal and tactile hyperalgesia.

Stress is antinociceptive in some models of pain, but enhances musculoskeletal nociceptive responses in mice and muscle pain in patients with fibromyalgia syndrome. To test the hypothesis that urocortins are stress hormones that are sufficient to enhance tactile and musculoskeletal hyperalgesia, von Frey fibre sensitivity and grip force after injection of corticotropin-releasing factor (CRF), urocortin I and urocortin II were measured in mice. Urocortin I (a CRF1 and CRF2 receptor ligand) produced hyperalgesia in both assays when injected intrathecally (i.t.) but not intracerebroventricularly, and only at a large dose when injected peripherally, suggesting a spinal action. Morphine inhibited urocortin I-induced changes in nociceptive responses in a dose-related fashion, confirming that changes in behaviour reflect hyperalgesia rather than weakness. No tolerance developed to the effect of urocortin I (i.t.) when injected repeatedly, consistent with a potential to enhance pain chronically. Tactile hyperalgesia was inhibited by NBI-35965, a CRF1 receptor antagonist, but not astressin 2B, a CRF2 receptor antagonist. However, while urocortin I-induced decreases in grip force were not observed when co-administered i.t. with either NBI-35965 or astressin 2B, they were even more sensitive to inhibition by astressin, a non-selective CRF receptor antagonist. Together these data indicate that urocortin I acts at CRF receptors in the mouse spinal cord to elicit a reproducible and persistent tactile (von Frey) and musculoskeletal (grip force) hyperalgesia. Urocortin I-induced hyperalgesia may serve as a screen for drugs that alleviate painful conditions that are exacerbated by stress.