Notes

The development of spongiosis is initiated by early keratinocyte apoptosis due to cell shrinkage and cleavage of E-cad, which is essential in mediating keratinocyte cohesion. Our data show that impairment and loss of keratinocyte cohesion constitute the primary event in spongiosis formation. Therefore, despite being the obvious driving force of spongiosis formation, fluid influx into the skin is apparently not the primary step, but rather the end result of a sequence of pathogenic events. Accordingly, dermal inflammation and intense fluid influx into the dermis in urticaria leave skin coherence totally intact . In contrast, in early lesions of bullous autoimmune skin diseases in which desmosomal cadherins are targeted by autoantibodies, spongiosis is visible It should be noted here that spongiosis is a nonspecific sign of cutaneous inflammation involving the epidermis. It is found in all kinds of eczemas, in bullous skin diseases, and in some viral and suferficial fungal infections as well. Spongiosis takes place mainly in the spinous layer of the epidermis. The heterogenous basal layer contains stem cells, transit amplifying cells, and postmitotic differentiating cells with high expression of integrins. It seems that in the basal layer at least stem cells exhibit strong antiapoptotic defenses In contrast to adherens junctions that may contain only E-cad, desmosomes always include cadherins from two subfamilies, Dsg and Dsc . We demonstrate that apoptosis-induced protein cleavage in keratinocytes is selective for certain adherens junction and desmosomal proteins. E-cad was cleaved, whereas b - catenin and desmosomal cadherins were not. The functional properties of E-cad and desmosomal cadherins are distinct despite their overall structural homology. The most striking sequence difference between Dsc, Dsg, and E-cad lies in their cytoplasmic tails . This may contribute to the selectivity of the cytoplasmic tails for different plaque proteins connecting them with different cytoskeletal Ælaments. These differences may also account for the differential behavior of desmosomal cadherins and E-cad in keratinocyte apoptosis. In the spongiotic epidermis of eczematous dermatitis, not all keratinocytes go into apoptosis. Therefore it is likely that in areas of intense spongiosis there is additional cleavage of cadherins on bystanding keratinocytes without ongoing apoptosis possibly due to proteinases released from secondary necrotic keratinocytes. The b -catenin binding domain of E-cad has been mapped to the residues 815±819 in the cytoplasmic tail (Miller and Moon, 1996). Cleavage of E-cad results in loss of coimmunoprecipitated b-catenin in this study. Recently, it was demonstrated that truncation of E-cad, loss of b -catenin binding, and cellular dissociation take place during prostate and mammary involution. In the same study E-cad mRNA was examined by northern blot and reverse transcriptase polymerase chain reaction and only the accumulation of a single transcript was found. Together these results demonstrate that loss of E-cad results from rapid, post-translational cleavage of the molecule, and not from transcriptional events. Similar E-cad truncation patterns by caspases were found in ischemic kidneys and apoptotic retinoblasts. E-cad acts as a substrate for activated caspases during keratinocyte apoptosis and its cleavage was inhibited by caspase inhibitors. These caspase inhibitors were also able to abrogate T-cell-induced keratinocyte apoptosis at the same concentration that blocked caspase-mediated cleavage events. Because of high levels of glycosylation of cadherins, several potential caspase cleavage sites, and different antibody epitopes, it is difÆcult to accurately determine the size of the proteolytic fragments by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The discrepancy be-tween theoretically predicted and observed molecular weights was reported previously. Proteolytic fragments of various sizes were observed (e.g., 97 kDa, 80 kDa, 48 kDa) in different cell types with different antibodies. We observed a cleavage product of about 85 kDa and this is consistent with proteolysis occurring at one o the potential caspase cleavage sites located towards the center of the molecule (Schmeiser and Grandt, 1999). Considering the molecular masses of the fragments and the antibody mapping, it was demonstrated that the cleavage site of E-cad during apoptosis is proximal to the transmembrane domain in the cytoplasm . At the 24 h time point the 85 kDa cleavage product was not consistently detectable, suggesting that further degradation
may also occur. In a human breast epithelial and a canine kidney cell line, apoptosis-induced cleavage of b-catenin as well as E-cad by caspases results in proteolytic fragments with reduced transactivation potential. During endothelial apoptosis cleavage of b-catenin and plakoglobin and shedding of vascular endothelial cadherin may act in concert to disrupt structural properties of adherens junctions. Accordingly, we examined how E-cad and b -catenin complexes were affected by the onset of apoptosis. We found loss of E-cad and b -catenin association but no obvious b-catenin degradation during keratinocyte apoptosis. In bovine pulmonary artery endothelial cells, lipopolysaccharide-induced apoptosis revealed cleavage of b - catenin and g -catenin, but vascular endothelial cadherin remained intact. These discrepancies in the observed fate of cadherins and b-catenin during apoptosis may be
partly due to the use of cell lines, e.g., in melanoma cell lines apoptosis pathways are changed and mutations in the b -catenin
gene were observed. Cadherin-mediated cell±cell contacts could substitute for integrin±extracellular matrix adhesion to prevent apoptosis in squamous cell carcinoma cells. In our experiments with primary human keratinocytes, functional blocking of E-cad alone did not induce keratinocyte apoptosis. The morphology of cultured keratinocytes in the presence of the blocking anti-E-cad MoAb, however, resembled the in vivo observed spongiosis. T cells inÆltrating the skin in eczematous dermatitis induce keratinocyte apoptosis. The early apoptotic response of keratinocytes is characterized by cleavage of E-cad, whereas desmosomal cadherins remain intact. Hydrostatic pressure, which is an important factor in the development of spongiosis, and the portions of the epidermal cell surface that still retain desmosomes may explain the elongation and distortion of remaining intercellular contacts observed in histopathology.