To further investigate the defects in the terminal keratinocyte differentiation induced by the lack of CerS3, a detailed analysis of SC ultrastructure was performed. While no intact peridermal cell layer could be observed in control epidermis (Figure 37A), the presence of this transitional layer was confirmed in neonatal CerS3 depleted epidermis (Figure 37B-F, P). A closer look at the peridermal architecture revealed the presence of desmosomes (Figure 37D and 37F, red arrows), as well as tight junctions (Figure 37F, red star). Therefore, no degradation, disaggregation or shedding of periderm cells was initiated neither at E18.5, nor at birth of CerS3 mutant
mice. Together with the hyperkeratotic phenotype of CerS3d/d, these findings might suggest a defect in the proper desquamation of mutant epidermis.
Consistent with this hypothesis, corneodesmosomes were found to persist in defective CerS3 epidermis. In mutant mice, corneodesmosomes remained abundant at all cell layers of the SC (supplemental Figure A5, red arrows), including the most superficial corneocyte layer (Figure 37B, black arrow), and were observed along the whole corneocyte surface. In contrast, corneodesmosomes were restricted to immature, inner corneocytes adjacent to the SG (stratum compactum) in control epidermis, and they were lacking at the uppermost SC layers (Figure 37A). As expected, the corneodesmosomal degradation as an important step of desquamation was initiated at the non-peripheral (basal and apical) surface of inner corneocytes and progressed also at the peripheral/lateral intercellular spaces concomitantly with cornification in control SC (data not shown).
The SC of mutant CerS3 mice occasionally exhibited a pair of thin electron-dense bands located within the intercellular space (ICS) parallel to the surface of the CE (Figure 37C, yellow arrows). In CerS3d/d mice, the space between the limits of the CE was much less than 13 nm, which would correspond to the typical ICS occupied by lipid lamellae (White et al., 1988). Instead these thin bands, which were visible to the uppermost corneocyte layer, seem to correspond to a phospholipid bilayer.
The defective corneodesmosomal degradation was further analyzed by immunolocalization of junctional proteins together with their total quantification by immunoblotting. To address whether the loss of CerS3 lead to an aberrant expression of desmosomal proteins, the transmembrane cadherin desmoglein 1/2, as well as the plaque proteins desmoplakin and plakoglobin were examined. Among these studies, the immunolocalization of desmoglein 1/2 exhibited a striking difference between mutant and control epidermis. Whereas the expression of this cadherin was restricted to desmosomes of keratinocytes within the SS and the SG in control mice (Figure 38A), mutant skin expressed desmoglein 1/2 at all the epidermal layers including the complete SC and the periderm (Figure 38B). Although the overall protein levels were found to be decreased in CerS3d/d mice (Figure 38G), its distribution decorating the complete periphery of corneocytes suggests a defective or delayed degradation of mutant corneodesmosomes.
Figure 37. Persistence of periderm and corneodesmosomes in newborn CerS3 depleted mice.
(A) Superficial corneocytes decorated with inclusion body of control mice lacking adjacent peridermal remmants or non-peripheral corneodesmosomes. A persistant peridermal layer (P) was observed in mutant CerS3 mice (B-F). Non-peripheral corneodesmosomes (black arrows) were present between peridermal cells and the most superficial corneocytes (B), as well as between the most superficial corneocytes and lower adjacent layers (C) of CerS3 depleted mice. Typical phospholipid bilayers appeared to be present in the intercellular space between corneocytes of mutant SC (C, yellow arrows). Intact periderm in mutant epidermis exhibited desmosomes (D and F, red arrows), as well as tight junctions (F, red star).
Epoxy-embedded sections were treated with tannic acid.
In contrast, the expression and concentration of the desmosomal plaque proteins, desmoplakin and plakoglobin, were found to be not significantly altered (Figure 38G).
As expected, desmoplakin expression was found to be restricted to the nucleated layers of the epidermis (Figure 38C-D). In addition, the epidermal localization of plakoglobin and the cytoskeletal keratins associated with desmosomal complexes, K1 and K10, were found similarly expressed in control and mutant CerS3 mice (data not shown).
Regarding the defective degradation of non-peripheral/central corneodesmosomes in mutants, our results indicate desmoglein 1/2 to be essential for corneocyte cohesions and responsible for persistent cohesions of periderm cells. In addition, these findings suggested an impaired proteolysis of the corneocyte plasma membrane and consequently a defective formation of a mature cornified cell envelope.
To examine whether the defective degradation of corneodesmosomes in mutant SC was caused by the reduced expression of proteolytic enzymes, levels of epidermal proteases of the kallikrein and the cathepsin family were analyzed. The total protein levels of either cathepsin D and KLK5 were found not significantly altered (Figure 38G). However, the epidermal distribution of cathepsin D in CerS3d/d mice remarkably differed from their control littermates. Whereas cathepsin D was found expressed in all the epidermal layers of control mice including the SC (Figure 38E), its expression was markedly decreased at the upper cell layers of the SG and the SC of mutant epidermis (Figure 38F). The reduced expression of this protease, which has been involved in the degradation of desmosomes during desquamation may account for the persistence of corneodesmosome remmants in mutant epidermis.
Figure 38. Impaired corneodesmosomal degradation in CerS3d/d epidermis.
(A) Desmoglein 1/2 is strongly expressed in the SS and SG of control mice. (B) In addition, the SC of mutant mice had a prominent staining at the periphery of all corneocyte layers. To assure that the lipid lamellae do not mask the epitope in
control mice, both preparations were subjected to mild alkaline treatment prior to immunolabelling. (C-D) No differences in desmoplakin distribution could be observed in control and mutant epidermis. (E) Cathepsin D distribution was localized at all epidermal cell layers. The uppermost granular keratinocytes displayed the strongest immunofluorescence. (F) In CerS3d/d mice, the strongest cathepsin D signal intensity was in the SS fading towards the SC. (G) Immunoblot analyses of desmosomal and tight junction associated proteins, as well as epidermal proteases. Blots are representative of at least 3 animals per group. β-actin was used to normalize protein levels.
Immunohistochemical analyses of tight junctional proteins were additionally performed in order to determine whether in addition to impaired degradation of the corneodesmosomal protein desmoglein 1/2, a defect in other types of cell junctional complexes existed. The immunolocalization of the tight junctional transmembrane protein claudin 1 and the cytoplasmic associated plaque protein cingulin was found within keratinocytes of the SS and the SG in both control and mutant epidermis (Figure 39A-B and 39C-D). Neither junctional proteins did show a significant difference in their signal intensity and main distribution pattern between control and mutant epidermis, however claudin 1 expression was distinctively and additionally observed in the periderm at the skin surface of mutant mice (Figure 39B, white stars) confirming the ultrstructural observations about the presence of intact tight junctions between peridermal cells (Figure 37F, red star).
Regarding the organization of TJs, cingulin expression in CerS3d/d mice exhibited conspicuous differences. Whereas typical zonulae occludentes were established in control epidermis appearing as continuous fluorescent linear structures at the plasma membrane of the upper cell layers of the SG, mutant epidermis displayed weaker fluorescent punctuated signals within the uppermost granular keratinocytes.
Figure 39. Distribution of tight junctions and associated proteins.
TJs were visualized with claudin 1 (A-B) and cingulin (C-D). No significant differences were observed in their distribution, however claudin 1 exhibited staining on the surface of mutant epidermis marking the periderm (white star). Cingulin expression exhibited a punctated distribution around keratinocytes of the SS and SG of CerS3d/d skin (D), whereas control skin exhibited linear distribution around the cell borders. A residual nuclear staining within some peridermal cells could also be detected on the surface of CerS3d/d skin (yellow star). (E) F-actin was mainly expressed in the nucleated layers of the epidermis in control mice CerS3d/d. (F) F-actin distribution differed to control by being also strongly expressed in the lower layers of the SC and progressively fading towards the upper corneocytes. Both control and mutant epidermis exhibited distinct punctated staining in the outer most corneocyte layer (yellow arrows).
The distribution pattern of the cytoskeletal protein F-actin, which is associated with TJs and adherens junctions, was additionally examined by immunolabeling. Besides the intense expression of F-actin at the cell-cell borders of all viable layers of the control epidermis (Figure 39E), its distribution in CerS3d/d mice additionally included the lower layers of the SC (Figure 39F) indicating a delay or a defect in the final cornification process. Furthermore, a pearl-like staining at the surface of both control and CerS3 deficient skin was observed. This distinct punctated expression of F-actin corresponds to the inclusion bodies observed at the ultrastructural level marking the uppermost corneocyte layer (Figure 36C-D and 37A-C).
Considered together, the findings obtained by the ultrastructural and the immunohistochemical analyses indicate an alteration of terminal keratinocyte differentiation particularly of the corneodesmosomal degradation upon deficiency of CerS3. The reduced expression of specific proteolytic enzymes might account totally or partially for the lack of processing of corneodesmosomes, and thereby conserving the intercellular cohesion leading to “hyper”keratosis and persistence of the periderm.
3.3.5. Impaired maturation of granular keratinocytes correlates with altered