Emeritus Faculty, Acad Council, Dermatology
The three areas of research are:
(1) defining the role of matrix metalloproteinases (MMPs) in connective tissue remodeling of the skin;
(2) defining the macromolecular structures and their interactions in the cutaneous basement membrane zone (BMZ); and
(3) developing methods for delivery of extracutaneous gene therapy in epidermolysis bullosa.
Matrix metalloproteinases are pivotal enzymes in connective tissue remodeling. The events in signal transduction that govern MMP expression and activity and expression of inhibitory proteins are crucial for understanding wound healing, tumorigenesis, and certain genetic diseases.
The cutaneous BMZ is a complex structure at the interface of the epidermis and dermis. The synthesis, secretion and organization of macromolecules of the BMZ involve regulatory events that dictate integrity of the skin, that are crucial for wound healing, and that can be aberrant in genetic diseases. Our laboratory focuses on discovery, cloning, sequencing, and creating gene therapy approaches for patients with hereditary forms of epidermolysis bullosa, a serious (potentially lethal) skin disease.
The development of topical drug delivery systems has recently gained significant interest due to the ease of administration and lesser risks of systemic toxicity. The development of these new technologies utilizes the properties of the structure and function of the skin. The stratum corneum plays the largest role in affecting drug permeation, as the corneocytes and lipid matrix in this layer effectively prevent the diffusion of large molecules. In this review, we introduce the structure and function of the skin as it relates to topical drug delivery.
View details for DOI 10.1111/j.1529-8019.2012.01460.x
View details for Web of Science ID 000300678300002
View details for PubMedID 22353152
View details for PubMedID 10344009
Psoralens are a class of pharmaceutical agents commonly used to treat several cutaneous disorders. When irradiated with a mode-locked titanium: sapphire (Ti:sapphire) laser tuned to 730 nm, an aqueous solution of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) emits blue light. The emission spectrum is centered at 452 nm and is identical to that obtained by one-photon excitation with UVA excitation, and its magnitude depends quadratically on the intensity of laser excitation. These results suggest that two-photon excitation occurs to a potentially photochemically active state. To estimate the two-photon absorption cross section, it was first necessary to measure the emission quantum yield of HMT using 365 nm excitation at room temperature that resulted in a value of 0.045 +/- 0.007. The two-photon absorption cross section of HMT at 730 nm is therefore estimated to be 20 x 10(-50) cm4 s (20 Göppert-Mayer). The excited-state photophysics and photochemistry of psoralens suggest potential applications to cutaneous phototherapy in diseases such as psoriasis and dystrophic epidermolysis bullosa.
View details for Web of Science ID A1997WD16800021
View details for PubMedID 9066288
Herlitz junctional epidermolysis bullosa is an autosomal recessive disorder characterized by generalized blistering at the lamina lucida of the cutaneous basement membrane. The monoclonal antibody GB3 has been used as a diagnostic probe because of its lack of reactivity in patient skin. The antigen recognized by GB3 has been identified as laminin-5, a glycoprotein consisting of three subunits (alpha 3, beta 3 and gamma 2). To identify the laminin-5 protein chain that contains the epitope recognized by GB3 and to determine if chain assembly is required for antibody recognition, we expressed a gamma 2 protein constructed from a full-length gamma 2 cDNA. Radioimmunoprecipitation of the culture medium from 293 cells revealed that both GB3 and anti-gamma 2 polyclonal antibodies were capable of directly precipitating recombinant gamma 2 without coprecipitation of other proteins. In immunodepletion experiments, each antibody removed most of the protein that was reactive with the other antibody. The epitope recognized by GB3 is present only when the complex is in the native conformation because GB3 reacted only with the non-reduced laminin-5, but not the reduced laminin-5 in immunoblots. Moreover, because GB3 reacted with laminin-5 of SCC25 cells (gamma 2 in the heterotrimer) but not recombinant gamma 2 in 293 cells (gamma 2 alone) during indirect immunofluorescence staining, this epitope may be dependent upon a less stable conformation of gamma 2. We conclude that GB3 recognizes the gamma 2 chain of laminin-5 and that the epitope is entirely contained in the native form of the gamma 2 chain.
View details for Web of Science ID A1995TC99600003
View details for PubMedID 7594636
Laminin-5 is a heterotrimer composed of alpha 3, beta 3, and gamma 2 chains, produced by keratinocytes and the human squamous cell carcinoma line (SCC-25), and is one of the candidate proteins for the genetic lesion in junctional epidermolysis bullosa. Two-dimensional SDS-polyacrylamide gel electrophoresis (first dimension, nonreducing conditions; second dimension, reducing conditions) revealed that the immunoprecipitated laminin-5 from a SCC-25 cell fraction consisted of alpha 3, beta 3, and gamma 2 monomers, a beta 3 gamma 2 heterodimer, and an alpha 3 beta 3 gamma 2 heterotrimer. The presence of the beta 3 gamma 2 heterodimer, but not heterodimers containing an alpha 3 chain and any of the other chains, was suggestive of assembly of laminin-5 proceeding from a beta 3 gamma 2 heterodimer to an alpha 3 beta 3 gamma 2 heterotrimer. We showed, by cotransfection experiments using full-length recombinant beta 3 and gamma 2 chains in a human cell line devoid of endogenous laminin-5, that stable heterodimers can be formed in the absence of alpha 3 chain expression. In the SCC-25 cell fraction, the alpha 3 monomer pool was the smallest of the monomers. Pulse-chase experiments using the cell fraction also indicated that the heterotrimer was assembled after a 10-min pulse and was nearly absent after a 24-h chase. These results are consistent with the synthesis of alpha 3 being limiting for heterotrimer assembly, with rapid association of the alpha 3 chain with beta 3 gamma 2 heterodimers to form complete heterotrimers. Treatment with tunicamycin reduced the size of each of the laminin-5 subunits, indicating that all chains are glycosylated, but that N-linked glycosylation is not necessary for chain assembly and secretion.
View details for Web of Science ID A1995RY90900042
View details for PubMedID 7559513
Type I collagenase plays an important role in both tumor metastasis and the remodeling of connective tissue in normal human skin, during wound healing, for example, and may participate in the pathophysiology of some dermatological diseases such as skin cancer and a chronic blistering disease, recessive dystrophic epidermolysis bullosa. In an effort specifically to inhibit collagenase expression, we have designed phosphorothioate antisense oligonucleotides, linked at the 5' ends with photoreactive 4'-(hydroxyethoxymethyl)-4,5',8-trimethyl-psoralen (HMT), and directed them against the 5' end of the collagenase mRNA. Two antisense-HMT molecules targeting a region overlapping the initiation codon were compared. Only one contained the HMT moiety targeting a 5'TpA on its complementary sense strand, and we observed greater than 50-fold improvement on the cross-linking of this antisense oligonucleotide to its target sequence after ultraviolet A (UVA) irradiation. Likewise, sequence complementary to the 5'TpA target was also required to demonstrate specific inhibition of in vitro translation of collagenase mRNA. Tissue culture experiments, conducted by incubation of collagenase-specific antisense-HMT oligonucleotides with fibroblasts in monolayer or in 3-dimensional dermal equivalents, showed lowered collagenase levels 24 h after UVA irradiation as compared to controls. Initial screening of antisense oligomers for specific hybridization and photo-cross-linking is a useful step in the design of antisense oligonucleotides, and allowed us to design an HMT-linked antisense phosphorothioate oligonucleotide that specifically inhibits the expression of fibroblastic collagenase.
View details for Web of Science ID A1995TA37000017
View details for PubMedID 7557028
Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. The most severe, dystrophic (scarring) forms of EB demonstrate blister formation below the cutaneous basement membrane at the level of the anchoring fibrils. Ultrastructural observations of altered anchoring fibrils and genetic linkage to the gene encoding type VII collagen (COL7A1), the major component of anchoring fibrils, have implicated COL7A1 as the candidate gene in the dystrophic forms of EB. We have recently cloned the entire cDNA and gene for human COL7A1, which has been mapped to 3p21. In this study, we describe mutations in four COL7A1 alleles in three patients with severe, mutilating recessive dystrophic EB (Hallopeau-Siemens type, HS-RDEB). Each of these mutations resulted in a premature termination codon (PTC) in the amino-terminal portion of COL7A1. One of the patients was a compound heterozygote for two different mutations. The heterozygous carriers showed an approximately 50% reduction in anchoring fibrils, yet were clinically unaffected. Premature termination codons in both alleles of COL7A1 may thus be a major underlying cause of the severe, recessive dystrophic forms of EB.
View details for Web of Science ID A1994NL71200022
View details for PubMedID 8088783
C-Jun is a cellular transcription factor that can control gene expression in response to treatment of cells with phorbol esters, growth factors, and expression of some oncogenes. The ability of c-Jun to catalyze the transcription of certain genes is controlled, in part, by changes in the phosphorylation state of specific amino acids in c-Jun. One of the major sites that is phosphorylated during signal response is Ser73. Here we show that substitution of a negatively charged aspartic acid residue at 73 constitutively increased transcriptional activity of c-Jun. The Asp73 substitution also enhanced its availability to bind to DNA in a whole cell extract without altering its intrinsic DNA binding activity since the intrinsic activity was unaltered for the c-Jun mutant proteins expressed in a bacterial system. The negatively charged Asp substitution may mimic the negative charge of a phosphorylated serine at 73. The substitution of an uncharged alanine at 73 resulted in lowered activities. The N-terminal end of c-Jun containing these substitutions was fused to the DNA-binding region of the bovine papilloma virus E2 protein, and was able to confer the same activation properties to the fusion protein at the heterologous E2 DNA-binding site. Ser73 lies in a region of c-Jun previously proposed to bind an uncharacterized inhibitor, perhaps related to a protein of approximately 17.5 kD that coprecipitates along with our c-Jun or the JunE2 fusion products.
View details for Web of Science ID A1994NH13200027
View details for PubMedID 8165146
Recessive dystrophic epidermolysis bullosa (RDEB) is a mutilating disease of the skin characterized by recurrent blistering and erosions that result from compromised integrity of the basement membrane zone. In this study, fibroblasts derived from the skin of RDEB patients were characterized for expression of the major metalloproteinases, particularly interstitial collagenase. Consistent with previous reports on increased collagenase protein levels in fibroblasts from some RDEB patients, we found that steady-state levels of collagenase mRNA were significantly increased in fibroblast strains derived from three of five RDEB patients compared to fibroblasts obtained from normal donors. Stromelysin mRNA was elevated in the same three fibroblast strains, whereas expression of neither the 72- nor the 92-kDa type IV collagenases was different from that of controls. Tissue inhibitor of metalloproteinases was expressed in RDEB fibroblasts at levels similar to those observed in normal fibroblasts. To investigate the mechanism behind the steady-state elevation in collagenase and stromelysin expression, AP-1 expression and activation were studied. Although levels of Jun expression were not different from those seen in normal fibroblasts, AP-1 activity, as assessed by ability to bind to a TPA response element-containing oligonucleotide, was endogenously elevated in RDEB fibroblasts compared to normal fibroblasts. Transfection studies using a plasmid construct containing the collagenase promoter linked to a CAT reporter gene demonstrated that RDEB fibroblasts were able to support active transcription of the promoter compared to normal fibroblasts. These studies support the hypothesis that RDEB fibroblasts contain chronically activated AP-1, and perhaps other transactivating factors, that contribute to the cellular phenotype of collagenase and stromelysin overexpression.
View details for Web of Science ID A1994NE89700006
View details for PubMedID 8143767
A variety of methods were used to fracture the dermal-epidermal junction (DEJ) of human skin. These included warm and hot phosphate buffered saline, trypsin, cold 1 M salt, potassium bromide and proteolytic digestion with dispase. The localization and sensitivity of basement membrane components (bullous pemphigoid antigen, BM 600/nicein, epiligrin, kalinin, laminin, collagens IV and VII (EBA antigen) and linkin) were determined after the DEJ was fractured by each method. We found that the basement membrane zone proteins, BM 600/nicein, epiligrin and kalinin remained with the dermal side of the DEJ fractured through the lamina lucida by cold salt, phosphate buffered saline and potassium bromide. BM 600/nicein, epiligrin and kalinin were not detected after treatment with trypsin. In contrast, laminin, another glycoprotein in the lamina lucida, was insensitive to all of the procedures, but co-localized to the dermal side of DEJ-fractured skin. We also found that separation of the DEJ with brief exposure of skin to 56 degrees C provided a useful substrate for testing the autoantibodies in the sera of patients with epidermolysis bullosa acquisita (EBA). Heat-separated skin can be prepared in a significantly shorter period of time than salt-separated skin.
View details for PubMedID 8357788
Blue-gray pigmentation of the skin can be elicited by several medications. We report the first case (to our knowledge) of desipramine-induced photosensitive blue-gray pigmentation.Diffuse blue-gray pigmentation on sun-exposed surfaces was noted in a healthy 48-year-old woman who had been taking desipramine hydrochloride for 8 years. Ultrastructural studies demonstrated the presence of melanin and homogeneous electron-dense material in the dermis.We conclude that tricyclic antidepressant agents represent another class of medications responsible for blue-gray cutaneous pigmentation.
View details for Web of Science ID A1993KW76500010
View details for PubMedID 8466219
Fibroblasts derived from the involved skin of scleroderma patients frequently display a phenotype of supernormal collagen expression when cultured. Fibroblasts displaying this phenotype derived from seven patients were treated with relaxin (1-100 ng/ml) and interferon-gamma (1-100 U/ml), individually and in combination, to assess the relative abilities of these cytokines to down-modulate collagen synthesis and secretion. Scleroderma fibroblasts displayed varying sensitivities to both relaxin and interferon-gamma. Relaxin (100 ng/ml) decreased expression of collagen by six of seven lines tested from 8 to 59% compared to untreated cultures. Interferon-gamma (100 U/ml) depressed collagen secretion by all seven lines in a range from 7 to 89%. When relaxin and interferon-gamma were used in combination, relaxin augmented IFN-gamma-induced decreases in collagen secretion in four of seven lines. In three of these lines, the use of relaxin in conjunction with suboptimal doses of interferon-gamma resulted in decreases equivalent to or greater than that seen with a tenfold higher concentration of interferon-gamma. This study demonstrates the ability of relaxin to directly alter the excessive collagen-producing phenotype of scleroderma fibroblasts. In addition, in some cases, combining relaxin and interferon-gamma resulted in a cooperative effect in decreasing collagen expression by scleroderma cells in vitro.
View details for Web of Science ID A1992JL54600016
View details for PubMedID 1512471
In order to identify structures in human skin that bind collagenase, sections from frozen or paraffin-embedded skin were incubated with either procollagenase or activated collagenase. After washing, bound procollagenase or collagenase was detected by immunofluorescence microscopy. In normal skin, procollagenase bound only to isolated granular dermal cells that were identified as mast cells on the basis of staining with fluoresceinated avidin and pinacyanol erythrosinate. When mast cells were degranulated by exposure to the ionophore A23187, extracellular granules bound procollagenase. Of various pathologic conditions examined, the highest binding of procollagenase occurred in specimens of urticaria pigmentosa. Procollagenase bound to granular cells and to abundant granules scattered throughout the dermis. Binding could be abolished by pre-treatment of tissue sections with heparinase or by pre-incubation of procollagenase with soluble heparin, suggesting that heparin is the binding agent in the granules. Activated collagenase also bound to dermal mast cells but in addition bound strongly to the dermal collagen. Enzymatic activity of activated collagenase was not inhibited by heparin in concentrations up to 10 mg/ml. There is evidence that mast cell tryptase can contribute to procollagenase activation. This study further supports a role for mast cells in collagenolysis by demonstrating that heparin from mast cells binds procollagenase and possibly serves as a reservoir for procollagenase, which may then subsequently be activated.
View details for Web of Science ID A1992HR03000014
View details for PubMedID 1373747
Recessive dystrophic epidermolysis bullosa (RDEB) is a subgroup of hereditary blistering diseases characterized by repetitive wounding and healing with subsequent extensive scarring. The purpose of this study was to establish a xenograft model that retains the RDEB phenotype and thus might be used as an experimental in vivo model to explore the molecular and biochemical mechanisms of the chronically wounded phenotype of RDEB. Full-thickness, tumor-free RDEB skin tissues were grafted onto the dorsum of severe combined immunodeficiency (SCID) mice. At 4, 8, 12, and 24 weeks after grafting, the xenografts were removed for examination. Immunofluorescence studies were performed using species-specific antibodies to human class I antigen, mouse class I antigen, human type IV and VII collagens and with cross-reacting antibody against bullous pemphigoid antigen (BPA). Staining with the antibody to human class I antigen, W6/32, and with the antibody to mouse class I antigen, 20.8.4s, confirmed the species-specific results obtained with the type IV and type VII collagen and laminin antibodies. The RDEB grafts showed essentially no staining with the type VII collagen antibody. Antibodies against laminin and BPA showed normal staining patterns in RDEB grafts. There was an overall paucity of anchoring fibrils in the grafts when examined with electron microscopy. Blisters could be induced in these grafts with minor trauma and showed a sublamina densa separation by immunomapping and electron microscopy. As late as 24 weeks post-transplantation, the RDEB grafts remain human, are not significantly replaced by mouse cells, and retain the RDEB disease phenotype.
View details for Web of Science ID A1992HB07100012
View details for PubMedID 1370678