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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02469

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The lysosomal cysteine protease cathepsin L regulates keratinocyte proliferation by control of growth factor recycling

¹ Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University, 79106 Freiburg, Germany
² Department of Biomedical Sciences, University of Bradford, Bradford, BD7 1DP, UK
³ German Cancer Research Center (Deutsches Krebsforschungszentrum), 69120 Heidelberg, Germany

View larger version (61K): [in a new window]   Fig. 1. Expression of murine cathepsin L (CTSL) in skin from the backs of ctsl+/+, ctsl-/- and Tg(K14-CTSL);ctsl-/- mice. (A) Transgene construct. Functional elements are the human keratin 14 promoter, the rabbit ß-globin intron, the full-length mouse CTSL cDNA, and the human growth hormone poly(A)signal. (B-D) Immunohistochemistry for detection of CTSL (brown staining; bars 20 µm). (B) Wild type; (C) Tg(K14-CTSL);ctsl-/-; and (D) ctsl-/-. (E) RT-PCR (endpoint amplification; 50 cycles) for detection of CTSL and GAPDH mRNA in primary dermal fibroblasts and epidermal keratinocytes. Lanes 1 and 8: 100 bp ladder; lanes 2 and 5: ctsl+/+; lanes 3 and 6: Tg(K14-CTSL);ctsl-/-; lanes 4 and 7: ctsl-/- (representative of three independent experiments). (F) Quantitative RT-PCR (real-time) for quantification of CTSL and GAPDH mRNA in liver, kidney and epidermis of wild type (ctsl+/+) and Tg(K14-CTSL);ctsl-/- mice (n=3). (F) CTSL proteolytic activity in epidermal lysates of wild type (ctsl+/+) and Tg(K14-CTSL);ctsl-/- mice (n=2).

View larger version (67K): [in a new window]   Fig. 2. Rescue of the ctsl-/- skin phenotype by keratinocyte-specific re-expression of CTSL. (A-C) Immunohistochemical detection of the proliferation marker Ki67 in basal keratinocytes (brown staining; bars 20 µm). (D) Quantitative analysis of epidermal thickness of mouse back skin (Axiovision; Zeiss). (E) Ki67 proliferation index (%) calculated as Ki67-positive basal layer cells divided by total basal layer cellsx100. ***P<0.001 compared with all other groups (n=3, four paraffin sections per mouse). (F,G) Rescue of the periodic hair loss of CTSL-deficient mice in Tg(K14-CTSL);ctsl-/- mice photographed 30 days after birth.

View larger version (68K): [in a new window]   Fig. 3. Mesenchymal-epidermal interactions assessed by heterologous organotypic co-cultures (OTC). Heterologous OTC consisting of ctsl+/+ fibroblasts or ctsl-/- fibroblasts in collagen type I gels topped by normal human primary keratinocytes were grown air-exposed for 7 days. Paraffin sections were stained in haematoxylin and eosin (HE) or by immunohistochemistry for the proliferation marker Ki67 (Ki67, brown nuclear staining) and the differentiation markers cytokeratin 10 (K10, brown staining) and transglutaminase (TG, brown staining). The experiment is representative for a total of 16 OTC grown with primary dermal fibroblasts from ctsl+/+ or ctsl-/- skin (two independent fibroblast preparations per genotype). Bars, 100 µm.

View larger version (25K): [in a new window]   Fig. 4. Paracrine and autocrine effects on keratinocyte proliferation in absence of CTSL. (A) Effects of fibroblast conditioned media on proliferation of ctsl+/+ (pale gray columns) and ctsl-/- keratinocytes (dark gray columns). Proliferation was measured by [3H]thymidine incorporation into DNA. *P<0.05 compared with all other groups. (B) Effects of keratinocyte conditioned media on proliferation of ctsl+/+ (pale gray columns) and ctsl-/- keratinocytes (dark gray columns). *P<0.05 compared with ctsl+/+; ***P<0.001 compared with all other groups.

View larger version (40K): [in a new window]   Fig. 5. CTSL-knockout keratinocytes are more responsive to EGF stimulation than wild-type cells. Proliferation of ctsl+/+ (pale gray columns) and ctsl-/- keratinocytes (dark gray columns) grown (A) in absence (no EGF) or (B) in presence (EGF) of exogenously added murine EGF (10 ng ml-1). The last supplementation with EGF was performed simultaneously with the addition of [3H]thymidine. *P<0.05 compared with ctsl+/+ keratinocytes. (C) Western blot for detection of EGF-receptor (EGFR) on keratinocytes and (D) quantitative RT-PCR (real-time) for quantification of EGF-receptor and GAPDH mRNA in absence of exogenous EGF.

View larger version (21K): [in a new window]   Fig. 6. Internalization of 125I-EGF by wild type (closed symbols) or CTSL-knockout (open symbols) primary keratinocytes. Cells were incubated with murine 125I-EGF for 30 minutes at 4°C. During the chase at 37°C, cell-surface-bound 125I-EGF (triangles) and intracellular 125I-EGF (squares) were quantified.

View larger version (28K): [in a new window]   Fig. 7. Enhanced recycling of 125I-EGF in ctsl-/- keratinocytes. After loading the cells for 30 minutes, cell-surface-bound 125I-EGF was removed by an isotonic pH 3.5 wash and the fate of the internalized 125I-EGF was followed during chase at 37°C. (A) Degradation of intracellular 125I-EGF in ctsl+/+ and ctsl-/- keratinocytes. (B) Degraded 125I-EGF in the medium. (C) Intact 125I-EGF in the medium. *P<0.05 between ctsl+/+ and ctsl-/- of the corresponding time point. (D) Cell surface (receptor-bound) 125I-EGF releasable by incubation at pH 3.5. *P<0.05, **P<0.01 between ctsl+/+ and ctsl-/- of the corresponding time point.

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