**3. Protease inhibitors**

Proteolytic activity in the skin, which is often restricted to a few target proteins, its tissue localization and its enzymatic activity, needs to be properly controlled in the tissue. Although gene expression and zymogen-activation are important regulatory elements to restrict enzymatic activity, the most important one is the expression of more or less specific protease inhibitors within the skin. These inhibitors regulate more or less proteasespecifically in a timely and concentration-dependent fashion the activity of diverse proteases. This review will summarize the current knowledge on the most important epithelial protease-inhibitors.

#### **3.1 Kazal-type-realted protease inhibitors**

The 'lympho-epithelial Kazal-type related inhibitor' (LEKTI, today named LEKTI-1) is an effective inhibitor of multiple serine proteases (Roelandt et al. 2009). Processing of this multidomain protease inhibitor into fragments or single domains restricts the inhibitory properties to serine proteases such as trypsin, plasmin, subtilisin A, cathepsin G and human neutrophil elastase. LEKTI-1 consists of 15 complete or incomplete Kazal domains. *In vitr*o, recombinant LEKTI-1 fragments or single domains inhibit the keratinocyte-derived serine proteases KLK5, -6, -7, -13 and -14. LEKTI-1 is expressed in various stratified epithelia as three splice variants. In the epidermis LEKTI-1 is expressed in the stratum granulosum, where LEKTI-1 protein is located in lamellar bodies – separate from KLKs, but secreted into the

Epidermal Serine Proteases and Their Inhibitors in Atopic Dermatitis 57

WAP-domain-containing proteins) (Schalkwijk et al. 1999). Human epidermis contains two, secretory leukocyte protease inhibitor (SLPI) and elafin. Both are efficient inhibitors of neutrophil serine proteases: SLPI inhibits cathepsin G and elastase, elafin inhibits elastase and protease-3. This suggests that these protease inhibitors are important at inflammatory conditions to protect the tissue from damage caused by neutrophil serine proteases. Whereas SLPI is constitutively expressed in the epidermis, elafin is present at a low level in healthy skin, but highly up-regulated at inflammatory conditions such as psoriasis (Wiedow et al. 1990). Elafin is stored in lamellar bodies and thus secreted as precursor at the interphase between stratum granulosum and stratum corneum, where it is crosslinked to

Another group of serine protease inhibitors are SERPINs, which encompass nearly 40 members, of which many have been implicated in cancer and inflammation (Meyer-Hoffert 2009). These protease inhibitors have a unique mechanism to inhibit enzymatic activity: SERPINs cause a conformational change of the protease and then covalently bind to it. A few members of the SERPIN family have been reported to be expressed in human skin, among them SERPINB3 (squamous cell carcinoma antigen-1), SERPINB4 (squamous cell carcinoma antigen-2), and SERPINB13 (headpin/hurpin). SERPINs have possibly a role in protecting tissue from proteolysis by bacterial proteases: SERPINB8 and SERPINB9 are inhibiting subtilisin A. SERPINA1 inactivates some microbial proteases including protease K. Further, a C-terminal fragment of SERPINA1 inhibits HIV-1 entry by

Apart from serine proteases also cysteine protease activity is under the control of inhibitors in skin: These include members of the cystatine gene family (Zeeuwen et al. 2009). Cystatins represent polypeptides which are members of a superfamily of evolutionarily-related proteins that can be divided in three subgroups, and which are widely expressed in several human tissues and secretions. They effectively inhibit various cysteine proteases, such as cathepsins B, L, H, K, and S, at micromolar to picomolar concentration in a competitive and reversible manner. Whereas cystatin A and cystatin C were reported to act as epidermal protease inhibitor with antimicrobial properties – possibly by inhibiting microbial cysteine proteases - cystatin M/E regulates in the epidermis crosslinking of structural proteins by transglutaminase 3 in the cornification process by controlling cathepsin L and legumain activities (Meyer-Hoffert 2009). Cathepsin L has been shown to activate transglutaminase 3, an epidermis-specific enzyme that is important in the cornification process where it is responsible for crosslinking of small proline-rich proteins and loricrin. A deregulation of this pathway by uncontrolled cysteine protease activity leads to abnormal stratum corneum

It should not be overlooked that the proteolytic activity of proteinases depends on factors like pH and ion-concentration. All serine proteases including KLKs decrease their proteolytic activity in acidic environments. The physiological pH of around 5.5 already results in more than 90% less active compared to optimal in vitro conditions. Patients with atopic dermatitis often show an elevated pH at the skin surface, which might likely contribute to observed elevated serine protease levels in these patients (Voegeli et al. 2009).

proteins of the CE via transglutamination.

interaction with the gp41 fusion protein.

and disturbance of skin barrier function (Zeeuwen et al. 2009).

**3.4 Other regulating factors of protease activity** 

**3.3 Cystatins** 

extracellular space together (Ishida-Yamamoto et al. 2004; Ishida-Yamamoto et al. 2005). The 145 kDa form comprises all 15 potential inhibitory Kazal-domains, but it is cleaved rapidly into multidomain fragments, which might be cleaved further to produce single domains and complexes with KLK5 and KLK7 in the SC. These complexes dissociate at acidic pH, which due to a pH gradient within the SC may lead to a controlled homeostatic desquamation. Mutations in *Spink5* (which encodes LEKTI-1) generating premature termination codons, as seen in Netherton Syndrome (Chavanas et al. 2000), result in expression of truncated LEKTI forms lacking several protease-inhibiting domains. This rare ichthyosiform skin disease is characterized by dry skin, increased desquamation, hair abnormalities ('bamboo hair') and atopy. A decreased level of functional LEKTI correlates inversely with serine protease activity in SC, a decrease physical barrier function and severity of the disease.

Another Kazal-type inhibitor is LEKTI-2 (*Spink9*), which has been originally discovered in palmar and plantar SC extracts (Brattsand et al. 2009; Meyer-Hoffert et al. 2009). LEKTI-2/SPINK9 is mainly expressed in palmar and plantar skin, close to KLK5. Apart from skin, expression was seen in the thymus (thus referred as LEKTI-2). All other tissues showed a very low transcription level of *Spink9*. LEKTI-2/SPINK9 selectively inhibited KLK5, but not other proteases including chymotryptic KLK7 and tryptic KLK14 or several serine proteases like trypsin and chymotrypsin. The LEKTI-2/SPINK9 activity differs in this respect from that of LEKTI-1: The Ki of LEKTI-2 was found in the range of 60 – 250 nM. LEKTI-1-domains have been reported to inhibit KLK5 in the range of 3 nM (domain 8-11) to 120 nM (domain 9-15). Further, the LEKTI-1 domains exhibit a more or less broad activity spectrum. It remains to be determined whether LEKTI-2/SPINK9 plays a role in skin diseases. Considering the specific expression of LEKTI-2/SPINK9 at palmar and plantar sites as well as its specific activity to inhibit KLK5, it is intriguing to speculate that it could be a relevant factor in hand and foot eczema.

By following the hypothesis that likely more Kazal-type inhibitors are present in human skin, we identified SPINK6 as a selective inhibitor of KLKs in the skin (Meyer-Hoffert et al. 2010). Unlike LEKTI-1 but similar to LEKTI-2, SPINK6 possesses only one typical Kazal domain. SPINK6 is strongly expressed, unlike LEKTI-2, in skin from various locations and can be purified from human plantar SC extracts. At low levels it is expressed in many other tissues and is induced during keratinocyte differentiation. While immunohistochemical analyses revealed SPINK6 expression in the stratum granulosum of healthy human skin at various anatomical localizations and in the skin appendages, including sebaceous glands and sweat glands, SPINK6 expression was found to be decreased in lesions of atopic dermatitis. Recombinant SPINK6 inhibited KLK4, KLK5, KLK6, KLK7, KLK12, KLK13 and KLK14 but not KLK1, KLK3 and KLK11, suggesting a tissue KLK-selective inhibitory activity, since thrombin, trypsin, plasmin, matriptase, prostasin, mast cell chymase, cathepsin G, neutrophil elastase, and chymotrypsin were not inhibited (Meyer-Hoffert et al. 2010; Kantyka et al. 2011). The finding that SPINK6 inhibited desquamation of human plantar callus in an *ex vivo* model suggests that SPINK6 plays a role in modulating the activity of KLKs in human skin. Interestingly, SPINK6 exhibited some proteolytic inhibitory activity against caspase-14 and is so far the only reversible inhibitor of caspase-14 in human skin (Kantyka et al. 2011).

#### **3.2 Trappins and serpins of human skin**

Apart from LEKTIs keratinocytes produce a number of additional protease inhibitors. Members of one group are termed 'trappins' (acronym for transglutaminase substrate, WAP-domain-containing proteins) (Schalkwijk et al. 1999). Human epidermis contains two, secretory leukocyte protease inhibitor (SLPI) and elafin. Both are efficient inhibitors of neutrophil serine proteases: SLPI inhibits cathepsin G and elastase, elafin inhibits elastase and protease-3. This suggests that these protease inhibitors are important at inflammatory conditions to protect the tissue from damage caused by neutrophil serine proteases. Whereas SLPI is constitutively expressed in the epidermis, elafin is present at a low level in healthy skin, but highly up-regulated at inflammatory conditions such as psoriasis (Wiedow et al. 1990). Elafin is stored in lamellar bodies and thus secreted as precursor at the interphase between stratum granulosum and stratum corneum, where it is crosslinked to proteins of the CE via transglutamination.

Another group of serine protease inhibitors are SERPINs, which encompass nearly 40 members, of which many have been implicated in cancer and inflammation (Meyer-Hoffert 2009). These protease inhibitors have a unique mechanism to inhibit enzymatic activity: SERPINs cause a conformational change of the protease and then covalently bind to it. A few members of the SERPIN family have been reported to be expressed in human skin, among them SERPINB3 (squamous cell carcinoma antigen-1), SERPINB4 (squamous cell carcinoma antigen-2), and SERPINB13 (headpin/hurpin). SERPINs have possibly a role in protecting tissue from proteolysis by bacterial proteases: SERPINB8 and SERPINB9 are inhibiting subtilisin A. SERPINA1 inactivates some microbial proteases including protease K. Further, a C-terminal fragment of SERPINA1 inhibits HIV-1 entry by interaction with the gp41 fusion protein.

#### **3.3 Cystatins**

56 Atopic Dermatitis – Disease Etiology and Clinical Management

extracellular space together (Ishida-Yamamoto et al. 2004; Ishida-Yamamoto et al. 2005). The 145 kDa form comprises all 15 potential inhibitory Kazal-domains, but it is cleaved rapidly into multidomain fragments, which might be cleaved further to produce single domains and complexes with KLK5 and KLK7 in the SC. These complexes dissociate at acidic pH, which due to a pH gradient within the SC may lead to a controlled homeostatic desquamation. Mutations in *Spink5* (which encodes LEKTI-1) generating premature termination codons, as seen in Netherton Syndrome (Chavanas et al. 2000), result in expression of truncated LEKTI forms lacking several protease-inhibiting domains. This rare ichthyosiform skin disease is characterized by dry skin, increased desquamation, hair abnormalities ('bamboo hair') and atopy. A decreased level of functional LEKTI correlates inversely with serine protease activity

Another Kazal-type inhibitor is LEKTI-2 (*Spink9*), which has been originally discovered in palmar and plantar SC extracts (Brattsand et al. 2009; Meyer-Hoffert et al. 2009). LEKTI-2/SPINK9 is mainly expressed in palmar and plantar skin, close to KLK5. Apart from skin, expression was seen in the thymus (thus referred as LEKTI-2). All other tissues showed a very low transcription level of *Spink9*. LEKTI-2/SPINK9 selectively inhibited KLK5, but not other proteases including chymotryptic KLK7 and tryptic KLK14 or several serine proteases like trypsin and chymotrypsin. The LEKTI-2/SPINK9 activity differs in this respect from that of LEKTI-1: The Ki of LEKTI-2 was found in the range of 60 – 250 nM. LEKTI-1-domains have been reported to inhibit KLK5 in the range of 3 nM (domain 8-11) to 120 nM (domain 9-15). Further, the LEKTI-1 domains exhibit a more or less broad activity spectrum. It remains to be determined whether LEKTI-2/SPINK9 plays a role in skin diseases. Considering the specific expression of LEKTI-2/SPINK9 at palmar and plantar sites as well as its specific activity to inhibit KLK5, it is intriguing to speculate that it could be a relevant

By following the hypothesis that likely more Kazal-type inhibitors are present in human skin, we identified SPINK6 as a selective inhibitor of KLKs in the skin (Meyer-Hoffert et al. 2010). Unlike LEKTI-1 but similar to LEKTI-2, SPINK6 possesses only one typical Kazal domain. SPINK6 is strongly expressed, unlike LEKTI-2, in skin from various locations and can be purified from human plantar SC extracts. At low levels it is expressed in many other tissues and is induced during keratinocyte differentiation. While immunohistochemical analyses revealed SPINK6 expression in the stratum granulosum of healthy human skin at various anatomical localizations and in the skin appendages, including sebaceous glands and sweat glands, SPINK6 expression was found to be decreased in lesions of atopic dermatitis. Recombinant SPINK6 inhibited KLK4, KLK5, KLK6, KLK7, KLK12, KLK13 and KLK14 but not KLK1, KLK3 and KLK11, suggesting a tissue KLK-selective inhibitory activity, since thrombin, trypsin, plasmin, matriptase, prostasin, mast cell chymase, cathepsin G, neutrophil elastase, and chymotrypsin were not inhibited (Meyer-Hoffert et al. 2010; Kantyka et al. 2011). The finding that SPINK6 inhibited desquamation of human plantar callus in an *ex vivo* model suggests that SPINK6 plays a role in modulating the activity of KLKs in human skin. Interestingly, SPINK6 exhibited some proteolytic inhibitory activity against caspase-14 and is so far the only reversible inhibitor of caspase-14 in human

Apart from LEKTIs keratinocytes produce a number of additional protease inhibitors. Members of one group are termed 'trappins' (acronym for transglutaminase substrate,

in SC, a decrease physical barrier function and severity of the disease.

factor in hand and foot eczema.

skin (Kantyka et al. 2011).

**3.2 Trappins and serpins of human skin** 

Apart from serine proteases also cysteine protease activity is under the control of inhibitors in skin: These include members of the cystatine gene family (Zeeuwen et al. 2009). Cystatins represent polypeptides which are members of a superfamily of evolutionarily-related proteins that can be divided in three subgroups, and which are widely expressed in several human tissues and secretions. They effectively inhibit various cysteine proteases, such as cathepsins B, L, H, K, and S, at micromolar to picomolar concentration in a competitive and reversible manner. Whereas cystatin A and cystatin C were reported to act as epidermal protease inhibitor with antimicrobial properties – possibly by inhibiting microbial cysteine proteases - cystatin M/E regulates in the epidermis crosslinking of structural proteins by transglutaminase 3 in the cornification process by controlling cathepsin L and legumain activities (Meyer-Hoffert 2009). Cathepsin L has been shown to activate transglutaminase 3, an epidermis-specific enzyme that is important in the cornification process where it is responsible for crosslinking of small proline-rich proteins and loricrin. A deregulation of this pathway by uncontrolled cysteine protease activity leads to abnormal stratum corneum and disturbance of skin barrier function (Zeeuwen et al. 2009).

#### **3.4 Other regulating factors of protease activity**

It should not be overlooked that the proteolytic activity of proteinases depends on factors like pH and ion-concentration. All serine proteases including KLKs decrease their proteolytic activity in acidic environments. The physiological pH of around 5.5 already results in more than 90% less active compared to optimal in vitro conditions. Patients with atopic dermatitis often show an elevated pH at the skin surface, which might likely contribute to observed elevated serine protease levels in these patients (Voegeli et al. 2009).

Epidermal Serine Proteases and Their Inhibitors in Atopic Dermatitis 59

be confirmed in two independent studies (Hubiche et al. 2007; Weidinger et al. 2008). *SPINK5* gene mutations are linked the with AD, when maternally inherited (Walley et al. 2001; Kato et al. 2003; Nishio et al. 2003; Weidinger et al. 2008). It is worth noting that the association was weaker than in the case of *FLG* mutations, in part, owing to a high prevalence in the control population. In a separate study on a French population, an association between *SPINK5* and AD was not found; however, there was an association between carriers and raised IgE serum levels (Hubiche et al. 2007)). The association of *SPINK5* mutations with raised IgE serum levels and with other atopic conditions, such as asthma, led to the suggestion that they are risk factors for general atopy (Walley et al. 2001; Nishio et al. 2003). In addition to *SPINK5*, a mutation has been identified in the *CSTA* gene encoding the cysteine protease inhibitor, cystatin A, which associates with AD. The cystatin A gene maps to chromosome 3q21, which has been identified as a major

Until today the complex pathogenesis of AD is not fully understood. But there is more and more evidence that epidermal proteases and their inhibitors are involved in the pathogenesis of AD. Their expression can be altered by genetic mutations and their activity is influenced by environmental factors like the pH, which is increased in AD patients (Sparavigna et al. 1999). Taken together there seems to exist a feedback regulation system important for skin barrier homeostasis in AD especially for kallikrein activity involving filaggrin mutations, PAR2 and LEKTI expression Tanaka 2011. It will be interesting to see whether inhibition of elevated protease activity will improve severity of AD lesions. Clinical trials are currently on their way with promising preliminary results

Part of this work was supported by grants of Deutsche Forschungsgemeinschaft (Me2037/3-1).

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Borgono, C. A., Michael, I. P., Komatsu, N., Jayakumar, A., Kapadia, R., Clayman, G. L.,

Brattsand, M., Stefansson, K., Hubiche, T., Nilsson, S. K. and Egelrud, T. (2009). "SPINK9: A Selective, Skin-Specific Kazal-Type Serine Protease Inhibitor." *J.Invest Dermatol.*  Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D. S.,

C. (2008). "Ichthyosis, Follicular Atrophoderma, and Hypotrichosis Caused by Mutations in ST14 Is Associated with Impaired Profilaggrin Processing." *J.Invest* 

Sotiropoulou, G. and Diamandis, E. P. (2007). "A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation." *J.Biol.Chem.* 282(6): 3640-

Weinrauch, Y. and Zychlinsky, A. (2004). "Neutrophil extracellular traps kill

susceptibility locus for AD (Lee et al. 2000).

**6. Conclusion** 

(http://clinicaltrial.gov).

**7. Acknowledgment** 

*Dermatol.* 

3652.

bacteria." *Science* 303(5663): 1532-1535.

**8. References** 

Interestingly, the inhibitory of activity of LEKTI-1 depends on the pH, too, which might enhance proteolytic deregulation, when the epidermal pH is elevated. Moreover, Zn2+ inhibits KLK5 (Debela et al. 2007). This might have clinical consequences, when Zink levels are low as in acrodermatits enteropathica. The exfoliation and inflammation observed in this disease might be a result of decreased KLK5 inhibitions.
