Skin barrier lipid enzyme activity in Netherton patients is associated with protease activity and ceramide abnormalities

Abstract

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum (SC) ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, β-glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) SC ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in SC ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered SC ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS. Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum (SC) ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, β-glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) SC ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in SC ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered SC ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS. activity-based probe acid sphing­omyelinase β-glucocerebrosidase 6-hexadecanoyl-4-methylumbelliferyl 6-hexadecanoyl-4-methylumbelliferylphosphorylcholine ichthyosis linearis circumflexa Netherton syndrome stratum corneum stratum granulosum Netherton syndrome (NTS) is a severe autosomal recessive disorder related to uncontrolled serine protease activity caused by mutations in the serine protease inhibitor Kazal-type 5 (SPINK5) gene that encodes for the protease inhibitor, lympho-epithelial Kazal-type-related inhibitor (LEKTI). This protein is crucial for proper skin desquamation (shedding of the skin). Increased epidermal serine protease activity in NTS patients results in scaling and superficial peeling of the skin and skin inflammation (1Greene S.L. Muller S.A. Netherton's syndrome. Report of a case and review of the literature.J. Am. Acad. Dermatol. 1985; 13: 329-337Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 2Traupe, H., 1989. The Ichthyoses: A Guide to Clinical Diagnosis, Genetic Counseling, and Therapy. Springer-Verlag, Berlin, New York.Google Scholar). Clinical manifestation varies to a high extent: some subjects demonstrate extensive and severe scaly erythroderma, whereas others develop ichthyosis linearis circumflexa (ILC) with variable severity. The origin for this variation is not fully understood (3Bitoun E. Chavanas S. Irvine A.D. Lonie L. Bodemer C. Paradisi M. Hamel-Teillac D. Ansai S. Mitsuhashi Y. Taieb A. et al.Netherton syndrome: disease expression and spectrum of SPINK5 mutations in 21 families.J. Invest. Dermatol. 2002; 118: 352-361Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Newborns are susceptible to life-threatening dehydration caused by increased water loss resulting from a defective skin barrier function (4Stoll C. Alembik Y. Tchomakov D. Messer J. Heid E. Boehm N. Calvas P. Hovnanian A. Severe hypernatremic dehydration in an infant with Netherton syndrome.Genet. Couns. 2001; 12: 237-243PubMed Google Scholar). This barrier is primarily located in the stratum corneum (SC) and formed by terminally differentiated keratinocytes (corneocytes) embedded in a lipid matrix (5Elias P.M. Menon G.K. Structural and lipid biochemical correlates of the epidermal permeability barrier.Adv. Lipid Res. 1991; 24: 1-26Crossref PubMed Google Scholar, 6Proksch E. Folster-Holst R. Jensen J.M. Skin barrier function, epidermal proliferation and differentiation in eczema.J. Dermatol. Sci. 2006; 43: 159-169Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). This matrix is composed of different lipid classes, like ceramides, cholesterol, and fatty acids. Whereas in other tissues ceramides are usually involved in metabolism or cell signaling, ceramides in the SC mainly function as skin barrier components. SC ceramides have very unique features compared with those present in other tissues: i) their carbon chains are much longer; and ii) there is a large variation in their molecular architecture (subclass overview in supplemental Fig. S1) (7van Smeden J. Bouwstra J.A. Stratum corneum lipids: their role for the skin barrier function in healthy subjects and atopic dermatitis patients.Curr. Probl. Dermatol. 2016; 49: 8-26Crossref PubMed Scopus (158) Google Scholar, 8Masukawa Y. Tsujimura H. Narita H. Liquid chromatography-mass spectrometry for comprehensive profiling of ceramide molecules in human hair.J. Lipid Res. 2006; 47: 1559-1571Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Ceramides are not synthesized in the SC, but their precursors are synthesized by keratinocytes located in the viable epidermal layers (9Holleran W.M. Takagi Y. Menon G.K. Legler G. Feingold K.R. Elias P.M. Processing of epidermal glucosylceramides is required for optimal mammalian cutaneous permeability barrier function.J. Clin. Invest. 1993; 91: 1656-1664Crossref PubMed Scopus (235) Google Scholar, 10Schmuth M. Man M.Q. Weber F. Gao W. Feingold K.R. Fritsch P. Elias P.M. Holleran W.M. Permeability barrier disorder in Niemann-Pick disease: sphingomyelin-ceramide processing required for normal barrier homeostasis.J. Invest. Dermatol. 2000; 115: 459-466Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). These ceramide precursors (glucosylceramides and sphingomyelins) are subsequently stored in lamellar bodies. These lamellar bodies also contain the enzymes necessary for the final conversion once the lamellar bodies extrude their content into the extracellular environment. This takes place at the interface of the viable epidermis, more specifically the stratum granulosum (SG), and the SC. The extruded ceramide precursors are then converted into their final barrier constituents by one final conversion step: sphingomyelins are converted into ceramides by acid sphingomyelinase (ASM; EC3.1.4.12), whereas glucosylceramides are converted by β-glucocerebrosidase (GBA; EC3.2.1.45). Importantly, GBA may convert glucosyl precursors of all ceramide subclasses, whereas ASM only converts sphingomyelin precursors into subclasses [AS] and [NS] (Fig. 1, supplemental Fig. S1) (11Hamanaka S. Hara M. Nishio H. Otsuka F. Suzuki A. Uchida Y. Human epidermal glucosylceramides are major precursors of stratum corneum ceramides.J. Invest. Dermatol. 2002; 119: 416-423Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 12Uchida Y. Hara M. Nishio H. Sidransky E. Inoue S. Otsuka F. Suzuki A. Elias P.M. Holleran W.M. Hamanaka S. Epidermal sphingomyelins are precursors for selected stratum corneum ceramides.J. Lipid Res. 2000; 41: 2071-2082Abstract Full Text Full Text PDF PubMed Google Scholar). Thus, conversion by ASM leads to ceramides with a sphingoid base only [see supplemental Fig. S1 for ceramide nomenclature (13Motta S. Monti M. Sesana S. Caputo R. Carelli S. Ghidoni R. Ceramide composition of the psoriatic scale.Biochim. Biophys. Acta. 1993; 1182: 147-151Crossref PubMed Scopus (352) Google Scholar)]. Previously, we reported an altered SC ceramide composition in NTS patients (14van Smeden J. Janssens M. Boiten W.A. van Drongelen V. Furio L. Vreeken R.J. Hovnanian A. Bouwstra J.A. Intercellular skin barrier lipid composition and organization in Netherton syndrome patients.J. Invest. Dermatol. 2014; 134: 1238-1245Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, it is unknown whether this change in ceramide composition is caused by a disbalance of epidermal GBA and ASM enzyme activities in the SC of NTS patients. In addition, the relation between expression/activity of both enzymes and how this relates to the ceramide composition and the clinical manifestation are not understood. Our aim was therefore to localize both expression and activity of ASM and GBA in the epidermis of 10 NTS patients (NTS1–10; denoted as circled numbers 1–10 in the table) and 5 healthy controls. The results provide mechanistic insight into how changes in localization of ASM and GBA associate with increased [AS] and [NS] SC ceramides, and whether this correlates with the localization of protease activity and patient clinical manifestations. The study was conducted according to Declaration of Helsinki principles, with written informed consent from patients (or parents in case of minors). Study approval was obtained from the Comité de Protection des in was at the de NTS patients in supplemental and supplemental Fig. compared with healthy controls. SC ceramides obtained by SC of the with 10 to ceramide for and in situ enzyme activity NTS patients, all from skin for in with matrix The and to 5 to enzyme for more and Fig. for an overview of the skin in with and at with for GBA and ASM in and with for GBA or ASM in and once in water and with A method was developed to visualize active ASM in human skin by in situ zymography 6-hexadecanoyl-4-methylumbelliferylphosphorylcholine as The are in the supplemental and skin in with ASM in with and in with with GBA was visualized the developed activity-based probe method Smeden J. H. A. S. J. et situ of in human skin zymography activity-based probe Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. van G. et in situ of active PubMed Scopus Google Scholar). skin for in in water for at with in once in and once in with with Skin of NTS patients embedded in at for 10 and with for 5 by two for 5 in Skin at with of 10 in 10 with for 5 and then with activity was visualized with a and with other a to a at of and of ASM was visualized by at GBA was visualized with at A was to the SC the This is the to SC the and the and J. M. A method for the and of from Full Text PDF PubMed Google Scholar, A method of lipid and J. PubMed Scopus Google Scholar). SC was with different of the of and with an of water and to from as the with and the in a of Full this method and are in W. S. Vreeken R. Bouwstra J. van Smeden J. of ceramides a with Biophys. Acta. 2016; PubMed Scopus Google Scholar). was by normal 5 to an with an of A was for in ceramide is as of areas two with A with a was to between the ceramide subclasses for both and the are with the We developed an in situ zymography method a ASM that results in the J. E. M. R. A enzyme for the of Niemann-Pick with of sphingomyelinase PubMed Scopus Google Scholar). in skin active ASM is localized at the interface between the and SC, as as the SC was also to a in more superficial SC layers and in the viable of are ASM activity in the lipid matrix the ASM activity is not the by the high in and that ASM is at the and to a large the results of the ASM activity However, the local variation in that was for ASM activity is not for the that not all ASM is In addition, ASM is also de in the viable epidermis the cell (Fig. ASM expression and activity in the epidermis of NTS patients large between subjects and a skin of all in supplemental Fig. NTS patients demonstrate the compared with i) areas with of active ASM also in the SC (Fig. ii) areas a or of active ASM at the interface compared with activity was located in the SC layers in some skin (Fig. or was Fig. iii) and ASM activity at the to skin (Fig. ASM two of expression in NTS skin i) areas that high expression of in of the SC (Fig. epidermal cell layers (Fig. extracellular ASM expression in areas was not at the interface but primarily as a layers the epidermis, the SC. ii) areas that expression at the areas a SC layers (Fig. We compared the GBA activity with the enzyme expression in NTS patients and controls. In active GBA is not in the or viable epidermis, but is the SC, with increased the interface (Fig. These of in the SC active GBA in the SC lipid matrix was not that active GBA is not present the SC. This in NTS and a large and between patients was In GBA activity was present at Fig. or a more with of active GBA located all epidermal layers (Fig. NTS skin with and a more normal GBA activity at the interface or all SC layers (Fig. GBA skin a at the interface and SC and there was expression in SC layers (Fig. GBA enzyme was also the of some epidermal particularly those to the In skin of NTS patients, was variable between and NTS subjects some skin areas in which GBA expression was to a expression at the interface (Fig. the other skin areas and the other NTS GBA expression in the the viable epidermis, with or a more expression at the and SC (Fig. This expression was particularly at skin areas with skin from NTS of patients demonstrate a expression activity skin 5 a of patients for the i) In areas with active GBA, active ASM was (Fig. with These areas a normal with GBA to ii) Skin areas with an a increase of active also the of localized in the lipid matrix (Fig. with iii) with a of active ASM not not also of active GBA and in supplemental Fig. In there was for an between the activity of GBA and than with the expression of both we compared the localization of the lipid enzymes with the localization of serine protease activity, not present in skin Fig. In NTS subjects a large variation in serine protease activity, as for the activity of ASM and areas with increased serine protease activity areas with ASM activity and GBA activity. This is for in Fig. of GBA and ASM the SC ceramide the ceramide as of the ceramide subclasses from SC of controls NTS patients in supplemental Fig. SC ceramide of was to an of lipid to that In controls a ceramide composition with ceramide the Smeden J. Janssens M. Bouwstra J.A. The role of stratum corneum for the cutaneous barrier Biophys. Acta. 2014; PubMed Scopus Google Scholar, Y. Narita H. H. A. N. Y. R. J. Takagi Y. et of ceramide in human stratum Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). NTS patients a in ceramide and a increase in ceramides [NS] and the only two ceramide subclasses that are of sphingomyelin by ASM (11Hamanaka S. Hara M. Nishio H. Otsuka F. Suzuki A. Uchida Y. Human epidermal glucosylceramides are major precursors of stratum corneum ceramides.J. Invest. Dermatol. 2002; 119: 416-423Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 12Uchida Y. Hara M. Nishio H. Sidransky E. Inoue S. Otsuka F. Suzuki A. Elias P.M. Holleran W.M. Hamanaka S. Epidermal sphingomyelins are precursors for selected stratum corneum ceramides.J. Lipid Res. 2000; 41: 2071-2082Abstract Full Text Full Text PDF PubMed Google Scholar). that the of subclasses NTS of ceramides [AS] and [NS] strongly correlated Fig. In addition, the of very ceramides in NTS patients (Fig. These ceramides only synthesized via GBA and are crucial for a proper skin barrier. an overview of the and the clinical for NTS The the for that GBA ASM expression not correlate with the clinical as scaly erythroderma or However, for activity to more in NTS patients with scaly erythroderma than in patients with activity was increased at areas with increased ASM activity and GBA activity. a relation between the clinical and the activity of GBA and ASM was the increase in the of SC ceramides [AS] and [NS] and the in ceramide more pronounced in patients with scaly erythroderma compared with patients with and not in patients with of of the and their to to was for the clinical and enzymes the ceramide and the protease activity. for expression and activity of lipid enzymes and protease was by skin changes changes or very different compared with skin of the SC ceramides are the of the for the not in a to was for the clinical and enzymes the ceramide and the protease activity. for expression and activity of lipid enzymes and protease was by skin changes changes or very different compared with skin of the SC ceramides are the of the for the not This study is the to and localize the expression and activity of both GBA and ASM in NTS. The zymography method that we This according to a more and than the J. E. M. R. A enzyme for the of Niemann-Pick with of sphingomyelinase PubMed Scopus Google Scholar, Man M.Q. D. Uchida Y. V. D. Feingold K.R. Elias P.M. serine activity by increase in leads to of lipid processing enzymes and of barrier function and stratum corneum Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). The developed method is also and enabled us to visualize with high active ASM in human with GBA activity labeling, we localize both active enzymes in the SC lipid layers of NTS patients and controls. This us to activity of key enzymes GBA and ASM to the ceramide NTS patients GBA expression and activity, particularly at areas with GBA was in NTS patients, but only active at the interface lipid and metabolism are crucial for optimal of the lamellar layers M. The epidermal lamellar a Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). A change in the local local or the of protein S. E. P. A. L. D. and for and for PubMed Scopus (33) Google Scholar, F. S. van C. R. van M. et of by active 12: PubMed Scopus Google may GBA to a in which GBA expression is by the cell to J. J. E. C. M. R.J. et activity in disease enzyme loss to with and Acad. Sci. PubMed Scopus (33) Google GBA in NTS The increase in ASM activity in the in subclasses [AS] and [NS] in as those ceramides are reported to the only subclasses that from conversion of sphingomyelins by ASM by (11Hamanaka S. Hara M. Nishio H. Otsuka F. Suzuki A. Uchida Y. Human epidermal glucosylceramides are major precursors of stratum corneum ceramides.J. Invest. Dermatol. 2002; 119: 416-423Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 12Uchida Y. Hara M. Nishio H. Sidransky E. Inoue S. Otsuka F. Suzuki A. Elias P.M. Holleran W.M. Hamanaka S. Epidermal sphingomyelins are precursors for selected stratum corneum ceramides.J. Lipid Res. 2000; 41: 2071-2082Abstract Full Text Full Text PDF PubMed Google Scholar). The between ceramides [NS] and [AS] between other supplemental S1) this is that ceramides [AS] and particularly [NS] function as molecules in case of or an R. and the sphingomyelin PubMed Scopus Google Scholar, of ceramide in to PubMed Scopus Google Scholar, J. and D. and Scholar). ASM is a key to increased activity in This an increase in [AS] and [NS] ceramides D. and of and acid PubMed Scopus Google Scholar, W. J. H. A. E. E. S. Y. et and other in and disease: metabolism and in and Biophys. Acta. 2006; PubMed Scopus Google and an increase in a for of and R. M. Y. Y. S. S. Y. et of sphingomyelin in the of the in human PubMed Scopus Google Scholar, M. and the J. Clin. Dermatol. PubMed Scopus Google Scholar, The role of sphingomyelin in of of cell differentiation and 2000; 13: Google Scholar). altered expression of ASM and levels of ceramides [AS] and [NS] are also related to skin like atopic dermatitis and (13Motta S. Monti M. Sesana S. Caputo R. Carelli S. Ghidoni R. Ceramide composition of the psoriatic scale.Biochim. Biophys. Acta. 1993; 1182: 147-151Crossref PubMed Scopus (352) Google Scholar, J.M. Folster-Holst R. A. M. S. C. S. E. sphingomyelinase activity and epidermal differentiation in atopic Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar, L. W. as in the in atopic J. 2014; PubMed Scopus Google Scholar, M. J. M. in inflammation for and skin J. Sci. PubMed Scopus Google Scholar). is reported that SC from a in ceramide to is in NTS These changes in the SC ceramide composition in NTS and other skin that the activity of GBA and ASM may also altered in other The that patients with scaly erythroderma and also demonstrate active ASM in the role of ASM as a of H. Ceramide and in the of PubMed Scopus Google Scholar). This is in with the of altered lamellar in NTS patients and in SC areas with M. Elias P.M. lamellar and stratum corneum in Netherton syndrome: differentiation from other and Dermatol. PubMed Scopus Google Scholar, C. C. M. Uchida Y. C. A. A. M. L. P. et is in human and epidermis and skin barrier function in Netherton syndrome and lipid Clin. Invest. PubMed Scopus Google Scholar, F. M. D. W. A. E. G. M. et protease activity and expression in Netherton Invest. Dermatol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In NTS the of related with the and of active i) SC areas very activity or active ASM to the interface to skin). ii) areas of very SC, as in and ASM activity key from this study is the between GBA, and serine protease NTS patients an extensive variation in the localization of active a in GBA activity coincided with an increase in both ASM activity and serine protease activity. for and this is at SC areas Fig. In the of serine protease activity correlated with the of active This a or between epidermal and lipid the local skin The of the SC and is crucial for epidermal barrier lipid enzyme function, and serine protease activity D. J. Feingold K.R. Elias P.M. epidermal permeability barrier and stratum corneum Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). in skin enzyme activity of lipid enzymes like GBA and which may to processed lamellar and a skin barrier Man M.Q. D. Uchida Y. V. D. Feingold K.R. Elias P.M. serine activity by increase in leads to of lipid processing enzymes and of barrier function and stratum corneum Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar). an increase in local skin in NTS to increased protease activity of 5 and the to These are involved in the desquamation and of lipid processing enzymes like GBA and ASM Man M.Q. D. Uchida Y. V. D. Feingold K.R. Elias P.M. serine activity by increase in leads to of lipid processing enzymes and of barrier function and stratum corneum Invest. Dermatol. Full Text Full Text PDF PubMed Scopus Google Scholar, and in the of atopic J. PubMed Scopus Google Scholar). NTS patients from defective which may to the defective skin barrier in patients F. M. D. W. A. E. G. M. et protease activity and expression in Netherton Invest. Dermatol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Hovnanian A. Netherton syndrome: defective in the skin leads to skin inflammation and 2014; PubMed Scopus Google Scholar). The increase in ceramides [AS] and [NS] to a more as with lipid in which ceramide subclasses increased Bouwstra J.A. New insight into and permeability of skin lipid and Biophys. PubMed Scopus Google Scholar). we the relation between the clinical of the 10 NTS subjects and the relation with SC and lipid between the expression of and the clinical was in with study in NTS (14van Smeden J. Janssens M. Boiten W.A. van Drongelen V. Furio L. Vreeken R.J. Hovnanian A. Bouwstra J.A. Intercellular skin barrier lipid composition and organization in Netherton syndrome patients.J. Invest. Dermatol. 2014; 134: 1238-1245Abstract Full Text Full Text PDF PubMed Scopus (57) Google and extensive but their expression that of healthy and with a of NTS but major in both GBA and ASM activity of both enzymes the clinical of all subjects very all subjects in activity compared with of NTS or skin and also displayed altered activity. localization of activity was with the severe of NTS with scaly erythroderma and These subjects also the levels of [AS] and [NS] In and a of displayed a ceramide composition that was all NTS to NTS patients as as other patients with skin that demonstrate changes in SC atopic whether the changes in SC ceramide subclasses and their enzyme and is a unique of NTS or a more for skin The of in this NTS study may for a of the disease and may in NTS in patients. of NTS or by and which may or to in which for with of Netherton Google Scholar). for SPINK5 mutations in not for of the ceramides and the enzyme expression/activity localization as a method that may in patients. the of a method to both and active ASM and GBA in situ enabled us to the relation between lipid the protease activity, and the SC ceramide composition in NTS patients. In addition, we demonstrate that in enzyme activities to the clinical of NTS. related to this at other are in the The and Boiten for in and des for The are to the for their

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