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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01075

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Cathepsin D is involved in the regulation of transglutaminase 1 and epidermal differentiation

¹ Department of Dermatology, University Hospital of Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany
² Institute of Immunology, University Hospital of Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany
³ Central Microscopy, Centrum for Biology, University of Kiel, 24118 Kiel, Germany
⁴ Department of Biochemistry, University of Kiel, 24118 Kiel, Germany

View larger version (22K): [in a new window]   Fig. 1. Increase in protein expression and activity of CTSD in differentiated keratinocyte cultures. (A) Protein expression of CTSD isoforms in the primary and differentiated keratinocytes was determined in cell lysates by western blotting using anti-CTSD antibodies. There was an increase in the prepro and enzymatically active pro forms in differentiated keratinocytes. (B) CTSD activity was measured by an in vitro enzyme assay of keratinocyte lysates using parathyroid hormone (PTH) as a CTSD-specific substrate. The amount of PTH in the absence of sample protein was used as a control. The level of PTH protein was determined by western blotting using anti-PTH mAb (peptide 1-34) and quantified by two-dimensional laser scanning densitometry (Molecular Dynamics Personal Densitometer). CTSD activity, calculated as the amount of PTH cleaved/hour, was increased in differentiated keratinocytes.

View larger version (25K): [in a new window]   Fig. 2. Increased epidermal expression of CTSD after experimental skin injury. Acute disruption of the permeability barrier was induced by tape-stripping. Immediately, pepstatin A or the carrier solution was applied and skin samples were obtained at different times. The expression of the active, intermediate (A) and the mature form (B) of CTSD were examined by SDS-PAGE and western blotting using polyclonal anti-CTSD antibody and quantified by two-dimensional laser scanning densitometry.

View larger version (32K): [in a new window]   Fig. 3. Increased activity of CTSD after experimental skin injury. Acute disruption of the permeability barrier was induced by tape-stripping. Immediately after barrier disruption, pepstatin A or the carrier solution was applied and skin samples were obtained directly after tape-stripping (0 hours) or after 3 and 5 hours. CTSD activity was measured by specific parathyroid hormone (PTH) enzyme assays. The level of PTH protein was determined by western blotting using anti-PTH mAb (peptide 1-34). The amount of PTH in the assay at the starting point was used as a control and CTSD activity was calculated as the amount of PTH cleaved/hour.

View larger version (15K): [in a new window]   Fig. 4. CTSD stimulates transglutaminase 1 (TG1) activity in primary keratinocytes in vitro. Isolated membranes from HaCat-cells were left untreated (black squares), treated with pepstatin A (black circles), or with purified CTSD in the absence (black triangles) or presence of pepstatin A (white triangles) for 30 minutes and were subsequently measured for TG1 enzymatic activity by an in vitro enzyme assay using dimethylcasein and [1,4(n)-3H] putrescine as substrates. Results of three experiments performed in triplicate are shown (mean±s.e.m.).

View larger version (17K): [in a new window]   Fig. 5. Topical application of the CTSD inhibitor pepstatin A (A) and of the TG inhibitor monodansyl cadaverin (MDC; B) significantly delays permeability barrier repair. Acute disruption of the permeability barrier was induced by tape-stripping until a 20- to 30-fold increase in TEWL (transepidermal water loss/transcutaneous water loss) occurred. Immediately after barrier disruption, pepstatin A, monodansyl cadaverin or the carrier solution was applied and recovery in TEWL was determined at different times after treatment. Results of three experiments performed in triplicates are shown.

View larger version (14K): [in a new window]   Fig. 6. Reduced transglutaminase 1 (TG1) activity in CTSD-deficient mice. TG1 enzymatic activity was determined in epidermal lysates from wild type, heterozygous and CTSD-deficient mice by an in-vitro assay using dimethylcasein and [1,4(n)-3H] putrescine as substrates. Results of three experiments performed in triplicate are shown (mean±s.e.m.).

View larger version (28K): [in a new window]   Fig. 7. Absence of 35 kDa TG1 protein in CTSD-deficient mice. TG1 protein levels were determined in epidermal samples from wild-type, heterozygous and homozygous CTSD-deficient mice by western blotting using an anti-TG1 antibody and quantified by densitometry.

View larger version (34K): [in a new window]   Fig. 8. Reduced CE protein expression in CTSD-deficient mice. Skin sections from wild-type, heterozygous and homozygous CTSD-deficient mice were obtained. Expression of involucrin (A) and loricrin (B) were determined in the epidermal samples by western blotting using anti-involucrin and anti-loricrin antibodies.

View larger version (118K): [in a new window]   Fig. 9. Immunohistology revealed distinct changes in the expression of keratins and CE proteins in CTSD-deficient mice. Keratin K1 staining in healthy skin is only found in suprabasal layers of the epidermis, whereas keratin K5 is only expressed in epidermal basal cells. In CTSD(–/–) mice there is a focal extension of K1 staining to the basal layer and a focal extension of K5 to the upper epidermal layers. Keratin K6 was faintly stained in normal mouse skin, but not in CTSD(+/–) or CTSD(–/–) mouse skin. Involucrin and filaggrin antibodies showed strong staining of the upper stratum spinosum and the stratum granulosum, whereas the loricrin antibody showed staining of the stratum granulosum solely in wild-type mouse skin. For all three antibodies staining intensity was reduced in CTSD(+/–) mice and even more reduced in CTSD(–/–) mice.

View larger version (97K): [in a new window]   Fig. 10. CTSD-deficient mice exhibited impaired stratum corneum morphology. Microscopic analysis of semi-thin skin sections from CTSD wild-type mice (A) revealed normal stratum corneum morphology. CTSD-deficient mice (B) have a disrupted stratum corneum and an increased number of corneocyte layers.

View larger version (94K): [in a new window]   Fig. 11. Ultrastructural changes of the stratum corneum and the transition of stratum granulosum to stratum corneum in CTSD(–/–) mice. Electron microscopy shows that in wild-type mice (A) the cornified envelope (CE) is clearly visible as dark lines around the corneocytes (arrow). In CTSD(–/–) mice (B) there is a broadening of the intercellular spaces in the stratum corneum (SC), only a faint staining of the CE (arrow) and the corneocytes are thickened in the axial direction. SG, stratum granulosum.

View larger version (41K): [in a new window]   Fig. 12. Model of the role of CTSD in epidermal differentiation. During epidermal differentiation, the aspartate protease CTSD in the stratum granulosum (SG) is activated, cleaves the membrane-bound 150 kDa precursor of transglutaminase 1 (TG1) producing the active 35 kDa form. TG1 in turn mediates cross-linking of the cornified envelope (CE) proteins involucrin and loricrin to the CE. SC, stratum corneum; SS, stratum spinosum; SB, stratum basale.