Η καλλικρεΐνη 6 (του ανθρώπου συμβολίζεται ως ΚLK6, ενώ του ποντικού ως Klk6), φαίνεται πως σχετίζεται με νευροεκφυλιστικές ασθένειες, όπως η σκλήρυνση κατά πλάκας και η νόσος του Parkinson, καθώς επίσης και με διάφορους τύπους καρκίνου. Επομένως, η μελέτη του ρόλου που διαδραματίζει η ΚLK6 στις παραπάνω ασθένειες σε πειραματικά πρότυπα ποντικού, μπορεί να χρησιμοποιηθεί για την κατανόηση αυτών των ασθενειών και στον άνθρωπο. Στην παρούσα εργασία αρχικά παρήχθη η ώριμη Κlk6 ποντικού ως ανασυνδυασμένη πρωτεΐνη στο σύστημα έκφρασης του μεθυλοτροφικού ζυμομύκητα Pichia pastoris KM71 και δείχθηκε ότι η παραχθείσα πρωτεΐνη ήταν ενζυμικά δραστική. Το cDNA που κωδικοποιεί την ώριμη Klk6 πρωτεΐνη απομονώθηκε με RT-PCR από τον εγκέφαλο ποντικού C57BL6. Το προϊόν πολλαπλασιάστηκε με αντίδραση PCR και μετά από πέψη με τα περιοριστικά ένζυμα XhoI και EcoRI κλωνοποιήθηκε στον φορέα pPIC9. Ο ζυμομύκητας Pichia pastoris KM71 μετασχηματίσθηκε με το ανασυνδυασμένο πλασμίδιο pPIC9/ώριμη Klk6 με τη μέθοδο των σφαιροπλαστών. Από τους ανασυνδυασμένους κλώνους του ζυμομύκητα που μετασχηματίσθηκαν με το πλασμίδιο έκφρασης απομονώθηκε πλασμιδικό DNA και η ορθή κλωνοποίηση και η απουσία μεταλλάξεων στην προς έκφραση αλληλουχία DNA επιβεβαιώθηκαν με αλληλούχηση. Στη συνέχεια, οι ανασυνδυασμένοι κλώνοι αναπτύχθηκαν σε υγρή καλλιέργεια και ελέχθηκε στο υπερκείμενο η παραγωγή ανασυνδυασμένης Klk6, δεδομένου ότι το σύστημα έκφρασης έχει σχεδιασθεί ώστε η ανασυνδυασμένη πρωτεΐνη να εκκρίνεται. Η ανασυνδυασμένη Klk6 απομονώθηκε από το υπερκείμενο και καθαρίσθηκε με χρωματογραφία υδρόφοβων αλληλεπιδράσεων ανοικτής στήλης. Μετά τον καθαρισμό, η απόδοση της παραγωγής ήταν 4,16 mg ανασυνδυασμένης Klk6 πρωτεΐνης ανά λίτρο (lt) αρχικής καλλιέργειας Η ενζυμική δραστικότητα της ανασυνδυασμένης Klk6 πρωτεΐνης ελέγχθηκε με ζυμογραφία ζελατίνης και καζεΐνης (ως υποστρώματα), καθώς και με βάση την ικανότητα υδρόλυσης χρωμογόνων συνθετικών πεπτιδικών υποστρωμάτων και πρωτεϊνικών υποστρωμάτων. Στη συνέχεια, πραγματοποιήθηκε η παραγωγή πολυκλωνικού αντισώματος ειδικού για την Klk6 πρωτεΐνη του ποντικού. Για την ανοσοποποίηση χρησιμοποιήθηκαν συνθετικά πεπτίδια με αλληλουχία που αντιστοιχεί σε δύο μη επικαλυπτόμενες περιοχές της Klk6, οι οποίες -κατά την υπολογιστική πρόβλεψη- χαρακτηρίζονται από υψηλή υδροφιλικότητα και αντιγονικότητα. Για την ανοσοποίηση, τον έλεγχο του τίτλου και τον καθορισμό των αντισωμάτων ακολουθήθηκαν καθιερωμένα πρωτόκολλα. Μετά τον καθαρισμό το κλάσμα των IgG αντισωμάτων αξιολογήθηκε ως προς την εξειδίκευση και τη συγγένεια έναντι της Klk6 ποντικού, χρησιμοποιώντας αρχικά την ανασυνδυασμένη Klk6 πρωτεΐνη, που παρήχθη όπως περιγράφηκε. Βρέθηκε ότι το παραχθέν πολυκλωνικό αντίσωμα αναγνωρίζει την πρωτεΐνη με υψηλή εξειδίκευση και ευαισθησία και το όριο ανίχνευσης προσδιορίσθηκε με ανοσοαποτύπωση πρωτεϊνών στα 50 ng. Τέλος, με RT-PCR αναλύθηκε το προφίλ έκφρασης της Klk6 σε ιστούς του ποντικού χρησιμοποιώντας εκκινητές ειδικούς για το γονίδιο Klk6. Χρησιμοποιώντας ολικά πρωτεϊνικά εκχυλίσματα από τους ιστούς εκείνους του ποντικού που βρέθηκε ότι εκφράζουν την Klk6, δείξαμε ότι το παραχθέν αντι-Klk6 πολυκλωνικό αντίσωμα αναγνωρίζει την ενδογενή Klk6 πρωτεΐνη με υψηλή ευαισθησία και ειδικότητα. Συνοπτικά, στην παρούσα εργασία παρήχθη με μεγάλη απόδοση ανασυνδυασμένη Klk6 πρωτεΐνη και με ενζυμικές δοκιμές δείχθηκε ότι είναι ενζυμικά δραστική, δηλαδή είχε την προβλεπόμενη πρωτεολυτική δράση. Επίσης, παρήχθη το πολυκλωνικό αντίσωμα αντι-Klk6 και δείχθηκε ότι αναγνωρίζει με υψηλή συγγένεια και ειδικότητα τόσο την ανασυνδυασμένη όσο και την ενδογενή Klk6 πρωτεΐνη σε ιστούς ποντικού και ως εκ τούτου θα αποτελέσει πολύτιμο εργαλείο για να μελετηθεί σε πρότυπα ποντικού ο ρόλος της πρωτεάσης Klk6 και η συμμετοχή της σε σηματοδοτικά μονοπάτια που σχετίζονται με σοβαρές ασθένειες, όπως η σκλήρυνση κατά πλάκας και η νόσος του Parkinson. / The kallikrein-related peptidase 6 (KLK6 for human; Klk6 for mouse) has been implicated in neurodegenerative diseases including multiple sclerosis and Parkinson’s disease and in various types of cancer. Therefore, the investigation of the role(s) of Klk6 in mouse models used for the study of the aforementioned human diseases becomes a necessity. First in this study, active recombinant mouse Klk6 protein was produced in Pichia pastoris KM71. The cDNA encoding for the mature mouse Klk6 protein was obtained by RT-PCR from RNA extracted from mouse C57BL6 brain. The PCR product was digested with XhoI and EcoRI, purified and subsequently cloned into the pPIC9 expression vector. The methylotrophic yeast strain Pichia Pastoris KM71 was transformed with the pPIC9/mature Klk6 construct, linearized with SalI, with the spheroplast method. Recombinant yeast clones transformed with the expression construct pPIC9/mature Klk6 were identified, confirmed by DNA sequencing and grown in liquid culture for recombinant protein production. Recombinant Klk6 was isolated from the yeast culture supernatant and was purified by hydrophobic interaction chromatography (HIC). The yield of recombinant protein production was 4.16 mg/lt culture after purification. Then, rKlk6 was characterized, in terms of proteolytic activity, by gelatin and casein zymography and by digestion of chromogenic synthetic peptide substrates and protein substrates. Νo antibody is currently available that is highly specific for mouse Klk6. Here, a novel rabbit anti-peptide polyclonal for mouse Klk6 was generated. Peptides corresponding to two non-overlapping Klk6 sequences of high hydrophilicity and predicted antigenicity were synthesized, conjugated to keyhole limpet hemocyanin (KLH) and used for immunization. The specificity and affinity of the purified polyclonal was assessed against active recombinant mouse Klk6 protein produced in Pichia pastoris KM71 as described, and the detection limit was determined at 50 ng by Western blot. Finally, the expression of Klk6 in mouse tissues was analyzed by semi-quantitative RT-PCR using gene-specific primers. Using whole extracts of Klk6 expressing mouse tissues, we showed that the purified anti-Klk6 polyclonal (IgG fraction) recognizes the endogenous Klk6 protein with a high sensitivity and specificity. Conclusively, the produced αντι-Klk6 polyclonal should be a valuable tool in studies employing mouse models in order to decipher the putative role(s) of Klk6 in signaling pathways involved in major human diseases, such as the multiple sclerosis and Parkinson’s disease.

Introduction. The outermost layer of the epidermis, the stratum corneum (SC), plays a fundamental role in our defense against microorganisms, chemicals, and dehydration. The SC is composed of tightly packed keratinized skin cells, corneocytes. For a functioning skin it is essential that corneocytes are constantly shed (desquamated). Kallikrein-related peptidase (KLK) 5 and KLK7 may be important in the desquamation process through degradation of desmosomal proteins. Severe hereditary diseases, where inhibition of KLK5 and/or KLK7 is missing, points to the importance of regulation of protease activity. KLKs may be regulated in various ways: tissue expression, activation of proforms, specific inhibitors, and physico-chemical properties like pH. Besides their involvement in desquamation, KLKs may also be important in immune defense and inflammation by processing of mediators and via activation of proteinase-activated receptors (PARs). Aims. 1. To identify and characterize previously unknown proteases in the SC. 2. To further characterize KLK5 and KLK7 with special focus on activation mechanisms. 3. To identify new inhibitors of KLKs in human SC. 4. To further characterize KLKs regarding effects of various inhibitors and substrates. 5. To study possible functions of KLKs in inflammation, in particular via activation of PAR-2. Methods. Plantar SC was used as a source for purification of proteins. Recombinant proteins were produced in different expression systems (insect cells, yeast cells, and bacteria). Different activity assays and kinetic studies were performed. Tissue expression was studied by immunohistochemistry, immunoblot and PCR. PAR-2 activation was studied by measurement of intracellular [Ca2+] and immunofluorescense in KNRK-PAR2 cells. Results. Active KLK14 was purified from extracts of plantar SC. KLK14 showed a superior catalytic efficiency as compared to KLK5 when measuring trypsin-like activity. This indicated that KLK14, despite being present in low amounts in skin, may have great relevance for skin physiology. Among enzymes tested only KLK5 showed autocatalytic activity and is so far the only enzyme found in SC that can activate proKLK7. KLK5 could also activate proKLK14. This together with studies of pH dependence on activation placed KLK5 as a possible key activating enzyme in a proposed proteolytic cascade in the SC. In plantar SC extracts we have also identified the novel Kazal-type serine protease inhibitor 9 (SPINK9). Our results indicate that SPINK9 is preferentially expressed in palmo-plantar skin and specific for KLK5. Differences found regarding substrate specificity and inhibition profile can be useful in evaluating the contribution of individual KLKs to the proteolytic activity in crude SC extracts. One interesting finding was that KLK8, present at high protein levels in the epidermis, could not be inhibited by any protease inhibitor found in the extracts. PAR-2 activation studies showed that KLK5 and 14 but neither KLK7 nor 8 can activate PAR-2. Immunohistochemistry preferentially detected KLK14 in intraepidermal parts of the sweat ducts and in dermal sweat glands but we could also show coexpression of KLK14 and PAR-2 in the SC and stratum granulosum of the epidermis in inflammatory skin disorders. To summarize, KLK involvement in desquamation may be dependent on a proteolytic activation cascade regulated by an intrinsic pH gradient and specific inhibitors present in SC. Another possible function of KLKs is as mediators of inflammation through activation of PAR-2.

Growing evidence indicates that serine proteases known as kallikreins are associated with malignancy and may have potential diagnostic/prognostic applications in cancer. Kallikreins are the largest group of serine proteases. Kallikrein enzymes are often involved in proteolytic cascades through their function in degradation of extracellular matrix proteins and promotion of angiogenesis. Kallikrein 6 (KLK6) is a member of the family of fifteen highly conserved secreted trypsin- or chemotrypsin-like serine proteases. Over-expression of KLK6 has been observed in different pathophysiological states such as neurodegenerative diseases, inflammation and various cancers, including colorectal cancer. In Chapter 3 we elucidated the miRNA-based mechanism of regulation of invasion in metastatic colorectal cancer over-expressing KLK6. We developed HCT116 colon stable isogenic cell lines with knockdown of KLK6 expression using short-hairpin interference RNA (shKLK6 clones). The shKLK6 clones had decreased expression and secretion of KLK6 protein with a minimal effect on cell growth and viability in cell culture. SCID mice injected with shKLK6-3 clone 3 cells exhibited a statistically significant increase in the survival rates (P=0.005), decrease in the incidence of distant metastases and a shift in the location of the metastatic foci closer to the cell's injection site. Levels of KLK6 protein secreted into the bloodstream were significantly lower in animals injected with shKLK6-3 clone 3 compared to HCT116 control clone 1 (P < 0.04). Through bioinformatics analyses we identified and validated three miRNAs, which are important in post-translational modification of bioactive proteins, proliferation, migration and p38 MAPK signaling pathway. In Chapter 4 we developed Caco-2 colon stable isogenic cell lines with expressing enzymatically active or mutant KLK6 protein (Caco-2 stable clones). We employed these cell lines to investigate the importance of KLK6 enzymatic activity of initiation of cell invasion using in vitro and in vivo models.

Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.