The Neuroendocrine Peptide Catestatin Is a Cutaneous Antimicrobial and Induced in the Skin after Injury Katherine A. Radek, Belen Lopez-Garcia, Melanie.

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The Neuroendocrine Peptide Catestatin Is a Cutaneous Antimicrobial and Induced in the Skin after Injury Katherine A. Radek, Belen Lopez-Garcia, Melanie Hupe, Ingrid R. Niesman, Peter M. Elias, Laurent Taupenot, Sushil K. Mahata, Daniel T. O'Connor, Richard L. Gallo Journal of Investigative Dermatology Volume 128, Issue 6, Pages 1525-1534 (July 2008) DOI: 10.1038/sj.jid.5701225 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Cst acts as an antimicrobial through membrane disruption. Membrane permeability was determined by measuring the release of β-galactosidase from E. coli ML-35p as detected by optical density (OD) 420nm. (a) A 1μgml−1 portion of polymixin B induces membrane leakage in the presence of 25mM NaHCO3 (open circles) but not in the absence of NaHCO3 (closed circles). A 25mM portion of NaHCO3 alone (open squares) had no effect compared to untreated cells (solid squares). (b) LL-37 (4μM, open circles) or Cst (20μM, open squares) induces membrane leakage in the presence of 25mM NaHCO3 but not in the absence of NaHCO3 (solid circles and squares). (c–h) Electron microscopy of E. coli 25922 treated for 10minutes with (c) 50μM Cst (original magnification × 40,000), (d) untreated (original magnification × 40,000), or 10minutes with (e) 32μM LL-37 (original magnification × 40,000). (f–h) Original magnification × 15,000 of E. coli shown in (c–e), respectively. Arrows in images (f–h) represent areas chosen for high magnification. Journal of Investigative Dermatology 2008 128, 1525-1534DOI: (10.1038/sj.jid.5701225) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Epidermal keratinocytes express Chga. Immunofluorescence analysis of Cst expression in human skin. Aldehyde-fixed sections of human skin were processed for immunohistochemistry using (a) a polyclonal anti-human Cst or (b) rabbit IgG. (c) Relative expression of Chga mRNA in human neuroblastoma SK-N-SH cells, whole human skin, cultured human keratinocytes, and cultured human fibroblasts using quantitative real-time PCR. Data represent the mean of three samples from the same tissue or cells normalized to GAPDH and then normalized to fibroblasts. Journal of Investigative Dermatology 2008 128, 1525-1534DOI: (10.1038/sj.jid.5701225) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 CHGA is processed into Cst in skin. (a) HPLC chromatogram of total human skin extract. The inset shows a peak at fraction 16 corresponding to the fraction indicated by the arrow that eluted at the same CH3CN% (28.5%) as human Cst synthetic peptide applied identically in a following run. Electrospray-ionization mass spectrometry identified the mass of this peak as 2,326Da. (b) Immunoreactivity of HPLC fractions shown in (a) as determined by reactivity to Cst antibody. Highest immunoreactivity is detected in fraction 16 indicated by the arrow. Journal of Investigative Dermatology 2008 128, 1525-1534DOI: (10.1038/sj.jid.5701225) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 CHGA is upregulated following barrier disruption and infection. Immunofluorescence analysis of Cst expression in injured mouse skin. Acetone-fixed sections of mouse skin subjected to consecutive tape-stripping +/- occlusion were processed for immunohistochemistry using a polyclonal anti-rodent Cst antibody. Cst staining is shown at (a) baseline, (b) 3hours post-tape stripping and (c) 3hours post-tape stripping with occlusion. (d) Rabbit IgG as a negative control. Bar=40μm. The expression of Camp (e) or Chga (f) was analyzed by quantitative PCR in normal, non-injured skin (control), or in skin 3hours after tape stripping +/- occlusion. The expression of Camp (g) or Chga (h) was analyzed by quantitative PCR in normal, non-lesional skin from uninfected mice (control), or in lesional skin 24hours after infection with GAS. Data are shown as the mean±SEM by Student's t-test, n=3–4 per group, repeated twice. *P<0.05. Journal of Investigative Dermatology 2008 128, 1525-1534DOI: (10.1038/sj.jid.5701225) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions