Kehua Li

Targeted Ablation of the Abcc6 Gene Results in Ectopic Mineralization of Connective Tissues

ABSTRACT Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6−/− mice but was not observed in Abcc6 +/− or Abcc6 +/+ mice up to 2 years of age. A total body computerized tomography scan of Abcc6 −/− mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease.

Molecular organization of the cutaneous basement membrane zone

Epithelial lesions of ulcerative, desquamative, and bullous character are major problems for clinicians and patients alike. Underlying such processes is often an immune or genetic cause in which the adhesion proteins responsible for maintaining the integrity of epithelium are affected. Autoimmune blistering skin diseases (ABSD) are progressive chronic debilitating diseases characterized by erosive lesions of the skin and/or mucous membranes. Several components of the epithelial basement membrane zone (BMZ) are targeted by the autoantibodies leading to cell detachment from the underlying tissue (subepithelial split). In recent years, efforts have been made to identify BMZ antigens recognized by autoantibodies in patients with various forms of ABSD. Depending on the primary site of involvement, autoantibodies may bind to distinct BMZ molecules: the bullous pemphigoid (BP) antigens of 230kD (BP230) or 180kD (BP180)1–4; the 3,5,6,7 3,8 and 29 subunits of laminin-5; the 4 integrin10; a 168– 170kD mucosal antigen11; type VII collagen,7,12; and 5 chain of type IV collagen.13 This chapter reviews the characteristics of the key molecules implicated in autoimmune diseases of the epithelium (Table 1).

Periplakin Gene Targeting Reveals a Constituent of the Cornified Cell Envelope Dispensable for Normal Mouse Development

ABSTRACT The members of the plakin family of proteins serve as epidermal cytolinkers and components of cell-cell and cell-matrix adhesion complexes, i.e., desmosomes and hemidesmosomes, respectively. Periplakin is a recently characterized member of this family. Human and mouse periplakin genomic loci are conserved, and the proteins are highly homologous, suggesting a role for periplakin in vertebrate physiology. In order to evaluate the functional role of periplakin, we generated periplakin null mice through targeted homologous recombination of mouse embryonic stem cells, followed by development of Ppl−/− mice. Mice homozygous for the targeted allele were born in the expected Mendelian frequency, developed normally, possessed grossly normal epidermis and hair, and were healthy and fertile. The epidermal barrier appeared to develop normally during fetal days E15.5 to E16.5, and the cornified envelope and desmosomes in the newborn mice were ultrastructurally normal. No compensatory increase in the expression of other epithelial proteins was detected in the neonatal mouse epidermis lacking periplakin. Consequently, the primary role of periplakin may not relate to the physiology of the cornified cell envelope in epidermal keratinocytes but may reside in the challenges, which normal laboratory mice do not encounter.

Cloning of the Gene for Human Pemphigus Vulgaris Antigen (Desmoglein 3), a Desmosomal Cadherin CHARACTERIZATION OF THE PROMOTER REGION AND IDENTIFICATION OF A KERATINOCYTE-SPECIFIC cis-ELEMENT

Pemphigus vulgaris antigen is a cadherin-like desmosomal cell adhesion molecule expressed primarily in suprabasal keratinocytes within the epidermis. Previously characterized structural features have defined this molecule as a desmoglein, DSG3. In this study, we have cloned the human DSG3 gene and examined the transcriptional regulation of its expression. The total gene consisted of 15 exons and was estimated to span >23 kilobases. Comparison of exon-intron organization of DSG3 with bovine DSG1 and several classical cadherin genes revealed striking conservation of the structure. Up to 2.8 kilobases of the upstream genomic sequences were sequenced and found to contain several putative cis-regulatory elements. The promoter region was GC-rich and TATA-less, similar to previously characterized mammalian cadherin promoters. The putative promoter region was subcloned into a vector containing chloramphenicol acetyl transferase reporter gene. Transient transfections with a series of deletion clones indicated that the DSG3 promoter demonstrated keratinocyte-specific expression, as compared with dermal fibroblasts examined in parallel, and fine mapping identified a 30-base pair segment at −200 to −170 capable of conferring epidermal specific expression. The results provide evidence for the transcriptional regulation of the pemphigus vulgaris antigen gene, potentially critical for development of the epidermis and physiologic terminal differentiation of keratinocytes.

Cloning of the mouse desmoglein 3 gene (Dsg3) : interspecies conservation within the cadherin superfamily

Abstract: Desmoglein 3 is a cadherin‐like calcium‐dependent cell adhesion molecule expressed primarily in suprabasal keratinocytes of the epidermis. In this study, we have cloned the full‐length cDNA and characterized the entire gene structure for the mouse desmoglein 3 gene (Dsg3). Isolation of overlapping cDNA clones, together with 5′ and 3′ rapid amplification of cDNA ends (RACE), allowed delineation of the entire coding sequence. The transcriptional initiation site was confirmed by primer extension and reverse transcription polymerase chain reaction analysis. The entire cDNA consisted of 6407 bp with an open reading frame of 2979 bp, and the deduced polypeptide contained 993 amino acids. Comparison of mouse and human desmoglein 3 amino acid sequences demonstrated 85.6% homology. Computer analysis suggested the presence of a transmembrane segment, 5 potential calcium binding sites, and a RAL motif which corresponds to the HAV motif, the potential site for homophilic interaction of typical cadherins. The mouse desmoglein 3 gene consisted of 15 exons in chromosome 18. Comparison of the intron–exon organization of Dsg3 with various cadherins from different species revealed remarkable conservation. This relatively high level of conservation both at the protein and genomic level suggests that desmoglein 3 plays an important role in keratinocyte cell–cell adhesion. Note

Bullous pemphigoid antigens (BPAGs): identification of RFLPs in human BPAG1 and BPAG2, and exclusion as candidate genes in a large kindred with dominant epidermolysis bullosa simplex.

Bullous pemphigoid antigens (BPAGs) are defined as autoantigens in a blistering skin disease, bullous pemphigoid. Two of the BPAGs, a 230-kD (BPAG1) and a 180-kD (BPAG2) protein, have been localized to hemidesmosomes, attachment structures at the basal keratinocyte-basement membrane interphase. We have recently cloned cDNAs corresponding to human BPAG1 and BPAG2, and mapped the corresponding genes to human chromosomes 6p and 10q, respectively. These cDNAs have now been used in a search for RFLPs in the corresponding genes. Southern hybridizations of genomic DNA from normal unrelated individuals with a BPAG1 cDNA detected an informative MspI RFLP, and similar hybridizations with a BPAG2 cDNA revealed an informative TaqI RFLP. These RFLPs were applied to a large kindred with epidermolysis bullosa simplex (EBS), generalized (Koebner) type, consisting of 14 affected and 12 unaffected individuals in three generations. Linkage analysis excluded the EBS locus in this pedigree approximately 9 cM and approximately 5 cM on either side of the BPAG1 and BPAG2 loci, respectively, when a lod score of -2.0 was taken as the limit of exclusion. This study demonstrates that mutations in the BPAG1 or BPAG2 genes are not the primary genetic defect in this family with EBS.

Expression of the Elastin Promoter in Novel Tissue Sites in Transgenic Mouse Embryos

We have previously shown in a transgenic mouse line, in which 5.2 kb of the elastin promoter was linked to the reporter enzyme chloramphenicol acetyltransferase (CAT), that the highest levels of expression were found in embryonic lungs and aorta, while lower levels were detected in other elastin-containing tissues. Furthermore, in general, expression of the transgene showed developmental regulation similar to that of the endogenous gene. However, the precise location of cellular expression could not be determined in this model. To overcome this limitation, we have developed a similar model, but replaced CAT with the reporter enzyme beta-galactosidase. Enzyme activity was readily detected in the transgenic mouse embryos in expected regions of tissue forming elastic fibers, including the dermis and elastic cartilage. Of considerable interest, however, was the novel finding of expression in specific areas of neuroepithelium of the brain and in the perichondrium surrounding areas destined to form hyaline cartilage in endochondral bone formation. These latter areas included all the bones of the limbs, the spine and rib cage. It appeared that these segments of elastin expression demarcated the border between the developing cartilage and the surrounding mesenchymal tissue. Elastin promoter expression was also found in developing somites, in the mesenchymal layer of the forming cornea of the eye, in the genital tubercle and in the epithelium destined to form the olfactory epithelium. These findings indicate that the elastin promoter is activated during embryonic development in a variety of tissues, suggesting that elastin gene expression may play a role in organizing cutaneous, skeletal and neural structures.