John F. Klement

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.

Mice deficient in involucrin, envoplakin, and periplakin have a defective epidermal barrier

The cornified envelope is assembled from transglutaminase cross-linked proteins and lipids in the outermost epidermal layers and is essential for skin barrier function. Involucrin, envoplakin, and periplakin form the protein scaffold on which the envelope assembles. To examine their combined function, we generated mice deficient in all three genes. The triple knockouts have delayed embryonic barrier formation and postnatal hyperkeratosis (abnormal accumulation of cornified cells) resulting from impaired desquamation. Cornified envelopes form but are ultrastructurally abnormal, with reduced lipid content and decreased mechanical integrity. Expression of proteases is reduced and the protease inhibitor, serpina1b, is highly upregulated, resulting in defective filaggrin processing and delayed degradation of desmoglein 1 and corneodesmosin. There is infiltration of CD4+ T cells and a reduction in resident γδ+ T cells, reminiscent of atopic dermatitis. Thus, combined loss of the cornified envelope proteins not only impairs the epidermal barrier, but also changes the composition of T cell subpopulations in the skin.

Type XIV Collagen Regulates Fibrillogenesis: PREMATURE COLLAGEN FIBRIL GROWTH AND TISSUE DYSFUNCTION IN NULL MICE*

Type XIV collagen is a fibril-associated collagen with an interrupted triple helix. This collagen interacts with the fibril surface and has been implicated as a regulator of fibrillogenesis; however, a specific role has not been elucidated. Functional roles for type XIV collagen were defined utilizing a new type XIV collagen-deficient mouse line. This line was produced using a conventional targeted knock-out approach. Col14a1(–/–) mice were devoid of type XIV collagen, whereas heterozygous mice had reduced synthesis. Both mutant Col14a1 genotypes were viable with a grossly normal phenotype; however, mature skin exhibited altered mechanical properties. Prior to evaluating tendon fibrillogenesis in type XIV collagen-deficient mice, the developmental expression patterns were analyzed in wild-type flexor digitorum longus (FDL) tendons. Analyses of mRNA and protein expression indicated tissue-specific temporal expression that was associated with the early stages in fibrillogenesis. Ultrastructural analyses of wild-type and null tendons demonstrated premature fibril growth and larger fibril diameters in tendons from null mice at postnatal day 4 (P4). However, fibril structure in mature tendons was normal. Biomechanical studies established a direct structure/function relationship with reduced strength in P7-null tendons. However, the biomechanical properties in P60 tendons were comparable in null and wild-type mice. Our results indicate a regulatory function for type XIV collagen in early stages of collagen fibrillogenesis with tissue differences.

Fibulin-2 Is Dispensable for Mouse Development and Elastic Fiber Formation

ABSTRACT Fibulin-2 is an extracellular matrix protein belonging to the five-member fibulin family, of which two members have been shown to play essential roles in elastic fiber formation during development. Fibulin-2 interacts with two major constituents of elastic fibers, tropoelastin and fibrillin-1, in vitro and localizes to elastic fibers in many tissues in vivo. The protein is prominently expressed during morphogenesis of the heart and aortic arch vessels and at early stages of cartilage development. To examine its role in vivo, we generated mice that do not express the fibulin-2 gene (Fbln2) through homologous recombination of embryonic stem cells. Unexpectedly, the fibulin-2-null mice were viable and fertile and did not display gross and anatomical abnormalities. Histological and ultrastructural analyses revealed that elastic fibers assembled normally in the absence of fibulin-2. No compensatory up-regulation of mRNAs for other fibulin members was detected in the aorta and skin tissue. However, in the fibulin-2 null aortae, fibulin-1 immunostaining was increased in the inner elastic lamina, where fibulin-2 preferentially localizes. The results demonstrate that fibulin-2 is not required for mouse development and elastic fiber formation and suggest possible functional redundancy between fibulin-1 and fibulin-2.

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.

Fibulin‐5 accelerates elastic fibre assembly in human skin fibroblasts

Abstract:  Fibulin‐5 null mice display abnormalities in the elastic fibres in the dermis. We postulated, therefore, that fibulin‐5 might be a regulator of elastic fibre assembly and stability. To clarify the role of fibulin‐5 in elastic fibre formation, we employed in vitro systems that allowed increasing expression of elastic fibre components by gene transduction using retroviral vector constructs. First, the human tropoelastin gene (ELN) was transduced into human dermal fibroblasts, which resulted in elevated gene expression. These cells were then cultured in monolayer, but the overexpression of ELN in this system did not alter the assembly of elastic fibres. However, incubation of fibroblasts with TGF‐β1 resulted in elastic fibre accumulation, and the expression of fibulin‐5 was enhanced by TGF‐β1. Thus, we overexpressed human fibulin‐5 cDNA in dermal fibroblasts using a retroviral vector containing a cytomegalovirus (CMV) promoter. These cells deposited elastic fibres. These results suggest that fibulin‐5 is a critical component in the control of elastic fibre assembly by dermal fibroblasts.

Targeted ablation of Abcc1 or Abcc3 in Abcc6−/− mice does not modify the ectopic mineralization process

Abstract:  Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by ectopic mineralization of connective tissues, with considerable intra‐ and interfamiliar phenotypic variability. PXE is caused by mutations in the ABCC6 gene, which encodes a transporter protein, MRP6, and targeted ablation of Abcc6 in mice recapitulates the manifestations of PXE. In this study, we examined the hypothesis that the expression of other members of the Abcc family may be altered in Abcc6 null mice, possibly explaining the phenotypic variability because of the functional overlap of these transporters. Analysis of the transcript levels of Abcc1–10 and 12 in the liver of Abcc6 −/− mice by quantitative RT‐PCR indicated that the levels of other C family mRNAs were not significantly different from wild‐type mice. Next, we developed Abcc6/1−/− and Abcc6/3−/− double null mice and examined them for tissue mineralization. Histopathologic examination, coupled with computerized morphometric analysis, and chemical assay of calcium × phosphate product in the muzzle skin of Abcc1−/− and Abcc3−/− mice did not reveal evidence of mineralization. Abcc6/1−/− and Abcc6/3−/− double knock‐out mice exhibited connective tissue mineralization similar to that in Abcc6 −/− mice. These results emphasize the importance of the Abcc6 gene in the ectopic mineralization process and further suggest that other members of the Abcc family, particularly Abcc1 and Abcc3, do not modulate the effects of Abcc6 in this mouse model.

Noninvasive Assessment of UV-induced Skin Damage: Comparison of Optical Measurements to Histology and MMP Expression

Acute exposure to UV radiation (UVR) causes visible skin damage such as erythema and results in local and systemic immunosuppression while chronic exposure can result in photocarcinogenesis. These deleterious effects can be quantified by histology and by bioassays of key biological markers, including matrix metalloproteinases (MMPs), or tryptophan moieties. We now report our results in quantifying UV skin damage with noninvasive optical methods based on reflectance and fluorescence spectroscopy and compare these noninvasive measurements to histopathology and MMP‐13 expression. A solar simulator with spectral output nearly identical to that of solar radiation was developed and used in our experiments. SKH1 hairless mice were exposed to solar‐simulated UVR at a total dose of 21 MED delivered over 10 weeks. Changes in oxygenated and deoxygenated hemoglobin were measured by diffuse reflectance spectroscopy, and tryptophan changes were monitored via a fluorescence monitor. Our results show that there is an increase in erythema, skin fluorescence, sunburn cells and MMP‐13 after a series of suberythemal doses of UV irradiation on a hairless mouse animal model. Increased skin fluorescence is observed with increasing UV exposure. The levels of MMP‐13 increase as the cumulative UV dose increases but their increase does not correspond to noninvasively measured changes.