Topical enzyme-replacement therapy restores transglutaminase 1 activity and corrects architecture of transglutaminase-1-deficient skin grafts

et al.Transglutaminase-1 (TG1)-deficient autosomal-recessive congenital ichthyosis (ARCI) is a rare and severe genetic skin disease caused by mutations in TGM1. It is characterized by collodion babies at birth, dramatically increased transepidermal water loss (TEWL), and lifelong pronounced scaling. The disease has a tremendous burden, including the problem of stigmatization. Currently, no therapy targeting the molecular cause is available, and the therapeutic situation is deplorable. In this study, we developed the basis for a causative therapy aiming at the delivery of the enzyme to the inner site of the keratinocytes’ plasma membrane. We prepared sterically stabilized liposomes with encapsulated recombinant human TG1 (rhTG1) and equipped with a highly cationic lipopeptide vector to mediate cellular uptake. The liposomes overcame the problems of insufficient cutaneous delivery and membrane penetration and provided excellent availability and activity of rhTG1 in primary keratinocytes. To demonstrate the general feasibility of this therapeutic approach in a humanized context, we used a skin-humanized mouse model. Treatment with rhTG1 liposomes resulted in considerable improvement of the ichthyosis phenotype and in normalization of the regenerated ARCI skin: in situ monitoring showed a restoration of TG1 activity, and cholesterol clefts vanished ultrastructurally. Measurement of TEWL revealed a restoration of epidermal barrier function. We regard this aspect as a major advance over available nonspecific approaches making use of, for example, retinoid creams. We conclude that this topical approach is a promising strategy for restoring epidermal integrity and barrier function and provides a causal cure for individuals with TG1 deficiency.This work was supported by the Bundesministerium für Bildung und Forschung as part of the Network for Ichthyosis and Related Keratinization Disorders (NIRK) (grants 01GM0901 and 01GM0902), the Foundation for Ichthyosis and Related Skin Types, and the Selbsthilfe Ichthyose e.V. F.L. was supported in part by the Instituto de Salud Carlos III (grant PI11/1225) and by grant S2010/BMD-2359 from C.M. M.D.R. was supported by the Ministerio de Ciencia y Innovación (grant SAF2010-16976). The work of M.D. was partially supported by Deutsche Forschungsgemeinschaft (grant DA 324/9-1).Peer reviewe

Topical Enzyme-Replacement Therapy Restores Transglutaminase 1 Activity and Corrects Architecture of Transglutaminase-1-Deficient Skin Grafts

Transglutaminase-1 (TG1)-deficient autosomal-recessive congenital ichthyosis (ARCI) is a rare and severe genetic skin disease caused by mutations in TGM1. It is characterized by collodion babies at birth, dramatically increased transepidermal water loss (TEWL), and lifelong pronounced scaling. The disease has a tremendous burden, including the problem of stigmatization. Currently, no therapy targeting the molecular cause is available, and the therapeutic situation is deplorable. In this study, we developed the basis for a causative therapy aiming at the delivery of the enzyme to the inner site of the keratinocytes’ plasma membrane. We prepared sterically stabilized liposomes with encapsulated recombinant human TG1 (rhTG1) and equipped with a highly cationic lipopeptide vector to mediate cellular uptake. The liposomes overcame the problems of insufficient cutaneous delivery and membrane penetration and provided excellent availability and activity of rhTG1 in primary keratinocytes. To demonstrate the general feasibility of this therapeutic approach in a humanized context, we used a skin-humanized mouse model. Treatment with rhTG1 liposomes resulted in considerable improvement of the ichthyosis phenotype and in normalization of the regenerated ARCI skin: in situ monitoring showed a restoration of TG1 activity, and cholesterol clefts vanished ultrastructurally. Measurement of TEWL revealed a restoration of epidermal barrier function. We regard this aspect as a major advance over available nonspecific approaches making use of, for example, retinoid creams. We conclude that this topical approach is a promising strategy for restoring epidermal integrity and barrier function and provides a causal cure for individuals with TG1 deficiency

The alpha and beta subunits of the metalloprotease meprin are expressed in separate layers of human epidermis, revealing different functions in keratinocyte proliferation and differentiation

The zinc endopeptidase meprin (EC 3.4.24.18) is expressed in brush border membranes of intestine and kidney tubules, intestinal leukocytes, and certain cancer cells, suggesting a role in epithelial differentiation and cell migration. Here we show by RT-PCR and immunoblotting that meprin is also expressed in human skin. As visualized by immunohistochemistry, the two meprin subunits are localized in separate cell layers of the human epidermis. Meprin alpha is expressed in the stratum basale, whereas meprin beta is found in cells of the stratum granulosum just beneath the stratum corneum. In hyperproliferative epidermis such as in psoriasis vulgaris, meprin alpha showed a marked shift of expression from the basal to the uppermost layers of the epidermis. The expression patterns suggest distinct functions for the two subunits in skin. This assumption is supported by diverse effects of recombinant meprin alpha and beta on human adult low-calcium high-temperature keratinocytes. Here, beta induced a dramatic change in cell morphology and reduced the cell number, indicating a function in terminal differentiation, whereas meprin alpha did not affect cell viability, and may play a role in basal keratinocyte proliferation

Long-term faithful recapitulation of transglutaminase 1-deficient lamellar ichthyosis in a skin-humanized mouse model, and insights from proteomic studies

This work was supported by the Bundesministerium für Bildung und Forschung as part of the Network for Rare Diseases NIRK (grant numbers: 01GM0901 and 01GM0902); the Foundation for Ichthyosis and Related Skin Types (F.I.R.S.T.); the National Institutes of Health (NIH) (grant number: P42 ES004699); and the Selbsthilfe Ichthyose e.V. FL was supported in part by the Instituto the Salud Carlos III (ISCIII) (grant number: PI081054), MDR was supported by the Ministerio de Ciencia y Innovación (MICINN) (grant number: SAF2010-16976). HCH was further supported by the Deutsche Forschungsgemeinschaft (DFG) (grant number: HE3119/5-1) and Köln Fortune (grant number: 79/2011

SPINK5 and netherton syndrome: Novel mutations, demonstration of missing LEKTI, and differential expression of transglutaminases

10.1111/j.0022-202X.2004.23220.xJournal of Investigative Dermatology1233474-483JIDE

CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

<div><p>We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.</p></div

Mitochondrial p27 is sufficient to induce endothelial cell migration.

<p><b>(A, B)</b> Endothelial cells were treated with 50 μM caffeine for 18 hours, and mitochondrial (“mito”) and nonmitochondrial (“non-mito”) fractions were separated. p27 and the closely related p21 protein were detected by immunoblot; TIM23 and Trx-1 served as purity controls for the fractions. <b>(A)</b> Representative immunoblots. <b>(B)</b> Semiquantitative analysis of mitochondrial p27 normalized to TIM23. Data are mean ± SEM, <i>n</i> = 6, *<i>p</i> < 0.05 (two-tailed unpaired <i>t</i> test). <b>(C)</b> Proteinase K digestion of mitochondria. The different digestion conditions yield intact mitochondria (1), mitochondria stripped of attached proteins (2), and mitoplasts (3); 4 denotes complete digestion. p27 and marker proteins for the outer (TOM40) or inner (TIM23) mitochondrial membrane and the mitochondrial matrix (GRP75) were detected by immunoblot. <b>(D, E)</b> Endothelial cells were transfected with an empty vector (“EV”) or expression vectors for nuclear (“nuc p27”) or mitochondrial p27 (“mito p27”). Expression and localization of the organelle-targeted p27 proteins were analyzed by immunoblot and immunofluorescence. <b>(D)</b> Representative immunoblot, Tubulin served as loading control. Because of the presence of a trimeric nuclear localization signal at the C-terminus, the nuclear-targeted protein has a larger molecular weight. <b>(E)</b> Representative immunostainings: nuclei were visualized with DAPI (blue), mitochondria by staining for TIM23 (red), and the targeted p27 variants by staining for the myc epitope (“myc (p27),” green). Merge shows an overlay of all fluorescence channels. <b>(F)</b> Endothelial cells were transfected with the siRNAs used in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.g001" target="_blank">Fig 1</a>. Forty-eight hours later, cells were transfected with an empty vector (“EV”) or the expression vectors for nuclear (“nuc p27”) or mitochondrial p27 (“mito p27”). Three hours later, a wound was set. Migratory capacity was assessed 18 hours later by counting cells migrated into the wound using Image J. Data are mean ± SEM, <i>n</i> = 5: p27, p27 siRNA-1/EV, p27 siRNA-2/EV, p27 siRNA-1/nuc p27 siRNA-1/mito p27; <i>n</i> = 6: all others, *<i>p</i> < 0.05 versus corresponding scr, ^#<i>p</i> < 0.05 versus scr/mito p27, ^§<i>p</i> < 0.05 versus p27 siRNA-1/mito p27, ^$<i>p</i> < 0.05 versus p27 siRNA-2/mito p27 (one-way ANOVA). <b>(G)</b> Endothelial cells were transfected with an empty vector (“EV”) or expression vectors for nuclear (“nuc p27”) or mitochondrial p27 (“mito p27”). Three hours later, a wound was set, and cells were treated with 50 μM caffeine for 18 hours or left untreated. Migratory capacity was assessed by counting cells migrated into the wound using Image J. Data are mean ± SEM, <i>n</i> = 5–7, *<i>p</i> < 0.05 versus EV −caffeine (Mann-Whitney pairwise comparison with Bonferroni-corrected <i>p</i>-values). <b>(H)</b> Endothelial cells were transfected with an empty vector (“EV”) or expression vectors for nuclear (“nuc p27”) or mitochondrial p27 (“mito p27”). Twenty-four hours after transfection, the mitochondrial membrane potential was measured with JC1 using flow cytometry. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus EV, ^#<i>p</i> < 0.05 versus nuc p27 (one-way ANOVA). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. DAPI, 4′,6-diamidino-2-phenylindole; HPF, high power field; n.s., not significant; TIM23, translocase of inner mitochondrial membrane 23; TOM40, translocase of outer mitochondrial membrane 40; Trx-1, thioredoxin-1.</p

Caffeine effects in the heart depend on p27.

<p><b>(A)</b> The mouse cardiomyocyte cell line HL-1 was lentivirally transduced with an empty vector (“EV”) or an expression vector for mitochondrially targeted p27 (“mito p27”) and treated with 500 μM H₂O₂ for 48 hours. Apoptosis was measured as annexin V positive/7-PI negative cells by flow cytometry. Data are mean ± SEM, <i>n</i> = 5, *<i>p</i> < 0.05 versus EV −H₂O₂, ^#<i>p</i> < 0.05 versus EV +H₂O₂ (one-way ANOVA). <b>(B)</b> Respiration was determined in isolated heart mitochondria of adult wild-type mice (“wt”) and p27-deficient littermates (“p27ko”), who had received drinking water without caffeine or water supplemented with 0.05% caffeine for 10 days. Respiration was measured as O₂ consumption without the addition of substrates (“mito”) and after the successive addition of malate/glutamate (“M/G”), ADP, rotenone (“rot”), and succinate (“succ”) (left panel). The right panel shows a magnification of O₂ consumption after the addition of M/G and ADP, respectively. Data are mean ± SEM, <i>n</i> = 5–8 per group, *<i>p</i> < 0.05 versus wt without caffeine (one-way ANOVA). <b>(C)</b> Adult p27-deficient animals and their wild-type littermates received drinking water or water supplemented with 0.05% caffeine for 10 days. RNAs were isolated from the hearts of those mice, and microarray analyses were conducted. Data are represented as a Venn diagram. The numbers in the circles indicate the number of transcripts regulated in the two genotypes (<i>n</i> = 3 animals per genotype and treatment, <i>p</i> < 0.05). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004408#pbio.2004408.s010" target="_blank">S1 Data</a>. ADP, adenosine diphosphate; n.s., not significant; PI, propidium iodide.</p