| Περίληψη: | Kallikrein-related peptidase 5 (KLK5 for human; Klk5, for mouse) or kallikrein 5 or stratum corneum tryptic enzyme (SCTE) is a serine protease and a pivotal regulator of skin desquamation, the physiological process of skin renewal (Brattsand et al., 2005; Borgoño et al., 2007). KLK5 is activated autocatalytically (Michael et al., 2005) and then, it activates other downstream proteases that are expressed in the epidermis, such as the KLK7 and KLK14 zymogens, to amplify the proteolytic activities that are engaged in cleavage of the outermost skin desmosomes. It is known that KLK5 hyperactivity is involved in pathological epidermal overdesquamation and inflammation associated with rare diseases, such as the Netherton syndrome (NS). In NS epidermis, genetic deficiency of the LEKTI inhibitor of serine proteases results in unopposed activity of KLK5 (and other serine proteases), which promotes skin inflammation via activation of the PAR2-NF-κB-TNFα signaling axis (Briot et al., 2009). In addition, proteolytic cleavage of cathelicidin by KLK5 results in production of antimicrobial and proinflammatory peptides (Yamasaki et al., 2006). Interestingly, the KLK5-PAR2-NF-κB-TNFα signaling pathway is activated and triggers inflammation also in common inflammatory skin disorders such as atopic dermatitis (AD) (Zhu et al., 2017; Elias PM, 2010).
PART I: Novel mouse models provide preclinical proof-of-concept for the combined use of KLK5 inhibitors and anti-TNFα therapeutics for effective treatment of Netherton syndrome
NS is an autosomal recessive multisystemic skin disorder that is characterized by severe epidermal overdesquamation and inflammation, infections, food and other allergies as a consequence of the extensive disruption of the skin barrier occasionally leading to neonatal lethality due to dehydration but also other systemic complications. NS is caused by mutations in SPINK5 (Serine Protease Inhibitor Kazal-Type) gene encoding the inhibitor LEKTI (Lympho Epithelial Kazal-Type-related Inhibitor) of serine proteases. LEKTI deficiency results in unopposed activities of serine proteases in the epidermis of NS patients. Spink5-/- mice recapitulate all major features of human NS, thus, providing a good model to study the disease. Spink5-/- mice are born apparently normal, nonetheless, within 30 min from birth they develop severe epidermal overdesquamation and inflammation and die uniformly 4-5 hours after birth due to extensive dehydration (Descargues et al., 2005). In a recent study published by our group in collaboration with Prof. Alain Hovnanian’s group, transgenic Spink5-/-Klk5-/- mice were generated to assess whether inhibition of KLK5 activity could be a pharmacological approach for NS. It should be noted that no specific therapy is currently available for NS. It was demonstrated that elimination of Klk5 in Lekti-deficient epidermis reverses the cutaneous hallmarks of NS in the early neonatal period (Furio et al., 2015). Spink5-/- mice have been generated by different groups, by differing transgenic approaches, and all show the same lethal phenotype (Yang et al., 2004; Descargues et al., 2005; Hewett et al., 2005; Kasparek et al., 2016). Here, Spink5-/-Klk5-/- mice were generated newly and monitored to evaluate whether inhibition of Klk5 could rescue NS for long periods. The Spink5-/- mice used in this study were provided by Prof. Andrew McKenzie and mimic the clinical features of NS (Hewett et al., 2005). We found that 70% of Spink5-/-Klk5-/- mice develop severe skin inflammation 5 to 7 days after birth triggered by activation of the Par2-NF-κB-Tnfα axis resulting in overexpression of the pro-Th2 cytokine Tslp and the proinflammatory cytokine Tnfα. Increased transcript levels of Il-1β, the Th17 type cytokine Il-17α and Il-23 were also observed in Spink5-/-Klk5-/- epidermis and strong infiltration by mast cells. In addition, aberrantly high proteolytic activities were detected in Spink5-/-Klk5-/- epidermis. Using several protease inhibitors with different substrate specificities a band corresponding to chymotrypsin-like activity was identified. By day 5-7, Spink5-/-Klk5-/- mice had developed a skin barrier defect as indicated by the toluidine blue dye penetration assay. The skin barrier defect is caused by the abnormally elevated proteolysis leading to enhanced cleavage of desmosomes and stratum corneum detachment, and secretion of the proinflammatory cytokine Il-1β by keratinocytes (Wood et al., 1992). Immunohistochemical staining showed reduced levels of the (corneo)desmosomal proteins Dsg1, Cdsn and Dsc1 and diffused staining likely a consequence of enhanced proteolytic cleavage. Further, decreased expression of involucrin and loricrin in Spink5-/-Klk5-/- epidermis indicated abnormal skin differentiation. Overall, we found that ~70% of Spink5-/-Klk5-/- mice die 5 to 7 days after birth due to severe skin overdesquamation and inflammation, which indicated that elimination of Klk5 alone is not sufficient to sustain a normalized skin barrier for long periods of time. A recent study showed that combined targeting of KLK5 and KLK7 proteases is indispensable for permanent restoration of the skin barrier defect in NS (Kasparek et al., 2016). Nonetheless, a major disadvantage of using cocktails of micromolecular inhibitors against KLK5 and KLK7 in humans is the current unavailability of specific inhibitors for KLK7, while KLK5 inhibitors are currently in development by Pharma (Personal communication; undisclosed). Thus, we investigated whether combined targeting of KLK5 and inflammatory pathways known to be involved in NS-associated inflammation could be an effective therapeutic approach. To provide proof-of-concept for such a pharmacological approach in humans, we have generated and characterized in depth the following novel preclinical mouse models: [1] Spink5-/-Klk5-/-Camp-/- and [2] Spink5-/-Klk5-/-Tnfα-/- mice in which, in addition to Klk5, the Camp gene (innate immunity), which encodes the cathelicidin precursor, and the Tnfa gene (adaptive immunity) were targeted, respectively. The percentage of mice that suffered postnatal lethality was not improved in Spink5-/-Klk5-/-Camp-/- mice, however, an increased lifespan was observed and some Spink5-/-Klk5-/-Camp-/- mice survived for 1 year, while none of the Spink5-/-Klk5-/- population survived longer than 8 months. Adult Spink5-/-Klk5-/-Camp-/- and Spink5-/-Klk5-/- mice exhibited epidermal overdesquamation and inflammation, and they died of an apparent skin barrier defect. Haemorrhagic areas were observed in the stomach of both genotypes in adult life but to a lesser extent in Spink5-/-Klk5-/-Camp-/-, however, the underlying molecular mechanisms were not investigated in terms of the present study. In Spink5-/-Klk5-/-Tnfa-/- mice, the skin barrier defect was fully reversed, no cutaneous abnormalities were observed macroscopically or microscopically, and no detached keratinocytes were identified in the surface of epidermis by scanning electron microscopy (SEM) and by hematoxylin/eosin staining of skin biopsies. Skin structure appeared normal with no detachment of the stratum corneum from the stratum granulosum, no hyperkeratosis or acanthosis. Furthermore, the expression levels of Dsg1, Cdsn and Dsc1 and of involucrin, loricrin and keratin 5 were normalized. Epidermal proteolytic activities were attenuated in Spink5-/-Klk5-/-Tnfa-/- and no skin inflammation was induced as reflected by the low numbers of infiltrating mast cells and lack of Par2 activation. Consistently, the cytokines Tslp, Il-1β, Il-17α, and Il-23 were not induced. Overall, 80% of Spink5-/-Klk5-/-Tnfa-/- mice have survived to adulthood. Adult Spink5-/-Klk5-/-Tnfα-/- mice sustain a normal skin microstructure with no inflammation or aberrant proteolysis and looked apparently like the wt mice except that they experience successive cycles of hair loss and regrowth. Permanent amelioration of the uniformly neonatal lethal phenotype of Spink5-/- mice by combined targeting of Klk5 and Tnfα is quite remarkable. It should be noted that anti-TNFα biologic factors are widely used in clinics as effective drugs for varying indications. Based on the described findings, we propose that the use of synthetic KLK5 inhibitors in combination with anti-TNFα biologic factors may represent a novel effective pharmacological approach for NS.
PART II: Investigating the role of KLK5 protease in skin cancer. Early skin tumorigenesis is suppressed in Klk5-deficient epidermis by reinforcement of the skin microstructure
KLK5 has been implicated in various types of cancer based on its aberrant expression in human tumors such as breast, testicular, renal and ovarian (Emani et al., 2008). However, not much is known on the underlying mechanisms. Considering that KLK5 is implicated in NS inflammation as elaborated above, we investigated whether KLK5 plays a role in skin cancer in vivo, since chronic inflammation is an important risk factor for the development of non-melanoma skin cancer. Skin carcinogenesis was induced in wt and Klk5-/- mice by application of DMBA/TPA or DMBA/DMBA according to established protocols. We show that Klk5-/- mice are resistant to early skin tumorigenesis. By contrast, the significantly fewer tumors that developed in Klk5-/- skin progressed faster to premalignant and malignant phenotypes. We have hypothesized that suppressed tumor formation in Klk5-deficient skin could reflect low inflammation because Par2-Nf-κb signaling is not activated. However, inflammation was much higher in Klk5-/- than in wt upon epicutaneous application of TPA. We have generated Tg-NglKlk5-/- mice to monitor Nf-κb activation in vivo. Specifically, Klk5-/- mice were intercrossed with Ngl transgenic mice carrying the firefly luciferase gene reporter, under the control of a minimal promoter carrying eight Nf-κb consensus sequences (Everhart et al., 2006). Against what we expected, strong activation of Nf-κb was observed in TPA-treated Klk5-/- skin which could not explain why early skin tumor formation is substantially suppressed in Klk5-/-. Thus, we investigated what could be the underlying molecular mechanism(s) that explain how and why Klk5-/- mice are resistant to early skin tumor formation. Since NF-κB activation in cancer cells is inhibited by β-catenin (Du et al., 2009; Deng et al., 2002), we studied the localization of β-catenin and found that, in Klk5-/- mice, β-catenin is not translocated into the nucleus upon TPA application but remains cytoplasmic, while in wt mice studied in parallel β-catenin was nuclear. Inhibition of β-catenin activation could explain the observed tumor suppression in Klk5-/- mice since it is known that β-catenin signaling promotes skin cancer development (Beronja et al., 2013). It should be noted that analysis by TEM of the epidermal ultrastructure revealed that desmosomes are significantly increased in numbers, length and electron density in Klk5-/- compared to wt mice. Thus, we hypothesized that immobilization of β-catenin in the cytoplasm could be associated with reinforced desmosomes in Klk5-/- skin and an enhanced mechanical strength. In fact, this was confirmed by AFM. Further, it is shown that inhibition of β-catenin results in induction of apoptosis as determined by TUNEL indicating that the number of tumors in Klk5-/- skin is likely reduced by apoptotic depletion of mutated keratinocytes. In summary, using Klk5-/- mice for the first time we provide evidence that KLK5 is implicated in early skin tumorigenesis in vivo by inhibiting oncogenic β-catenin signaling. Cumulatively, the present doctoral thesis aims to enhance our understanding of the molecular mechanisms implicated in skin inflammation and the role of Κlk5 protease, and to aid the future development of new effective therapies. The study provides proof-of-concept for the design of innovative therapeutic approaches for overdesquamating inflammatory skin diseases such as NS, but also for more common diseases including atopic dermatitis (AD) that share the underlying molecular mechanisms. Furthermore, the study provides evidence that KLK5 is implicated in early stages of skin tumorigenesis.
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