Hominoid primate species differ remarkably in their social grouping and mating systems, notably including differing degrees of post-copulatory sexual selection. As the mating system of extinct hominins remains unknown and difficult to predict, it may be useful to examine more proximate phenotypes correlated with behavior. For example, chimpanzees and bonobos have a large ejaculate that coagulates into a rigid copulatory plug, presumably in response to high levels of sperm competition, while gorillas have a small semi-viscous ejaculate associated with low sperm competition. To understand the molecular basis responsible for differences in semen biochemistry among hominoid species, I completed two research projects. First, by cloning the upstream putative promoters of the chimpanzee, bonobo, human, and gorilla prostatic acid phosphatase (ACPP) genes into luciferase reporter vectors followed by transient transfections into a human prostate cell line, I identified the underlying nucleotide changes that reduce expression of this protein in chimpanzee semen. Second, by mapping large deletions at the kallikrein-related peptidase (KLK) locus in the gorilla and gibbon genomes, I characterized the convergent gene loss and the formation of a novel chimeric gene in these monandrous species. For both the ACPP and KLK locus changes, I determined the polarity of the changes through outgroup comparison. At ACPP, the reduced expression in chimpanzee and bonobo is derived, and likely in response to the onset of intense sperm competition in the common ancestor of these two species. If this biochemical phenotype is indeed a proxy for mating behavior, my data provides some evidence (to be compared and contrasted with other molecular, behavioral, and paleontological data) that the last common ancestor of humans and chimpanzees was not chimp-like in its high degree of polyandry. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;

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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.

Kallikrein-related peptidases (KLKs) are a subgroup of serine proteases of undisputable importance for a variety of functions, whose dysregulation has been linked to several pathological phenotypes. Among those pathologies, the Netherton syndrome stands out, since it is one of the very few that has its mechanism directly linked to KLK proteases as the main culprit of the disease, namely KLK5, KLK7 and to a lesser degree, KLK14. In this case, a mutation in the SPINK5 gene leads to uncontrolled hyperactivity of those proteases, which results in epidermal barrier breach due to excessive epidermal desquamation and severe inflammation of the skin. Inflammation mechanisms of NS are still relatively poorly understood, with important roles being attributed to the activities of KLKs in the processing of immune system molecules and also to the dysregulation of the cutaneous microbiome. TNFα signalling plays a key role in the homeostasis and immune response in the skin. Chronic skin infections may lead to deleterious effects with strong participation of TNFα signalling. To address the degree of its effects on the pathogenesis of NS, we have created a mouse model where the TNFR1 is disrupted by knockout of the Tnfr1 gene on the background of a previously established mouse model of the Netherton syndrome. We...

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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.

At present, prostate cancer is a significant public health issue throughout the world and is the second leading cause of cancer deaths in older men. The prostate specific antigen or PSA (which is encoded by the kallikrein 3/KLK3 gene) test is the current most valuable tool for the diagnosis and management of prostate cancer. However, it is insufficiently sensitive and specific for early diagnosis, for staging of prostate cancer or for discriminating between benign prostatic hyperplasia (BPH) and prostate cancer. Recent research has revealed another potential tumour marker, glandular kallikrein 2 (KLK2 gene/hK2 protein), which may be used alone or in conjunction with PSA to overcome some of the limitations of the PSA test. Twelve new kallikrein gene family members have been recently identified and, like hK2 and PSA, many of these genes have been suggested to be involved in carcinogenesis. In this study, the cellular localisation and level of expression of several of these newer kallikreins (KLK4, KLK5, KLK7, KLK8 and KLK11) was examined in prostate tissue, to provide an understanding of the association of their expression with prostatic diseases and their potential as additional biomarkers. Like PSA and hK2, the present observation indicated that each of these proteins, hK4, hK5, hK7, hK8 and hK11, was detected within the cytoplasm of the secretory cells of the prostate glands. For the first time, all of these newly-identified proteins were shown to be expressed in prostatic intraepithelial neoplasia (PIN) lesions, in comparison to normal glands and cancer lesions. In addition to cytoplasmic secretory cell expression, the localisation of hK4 to the basal cells and nuclei in prostatic lesions was intriguing. The intensity of hK4 staining in prostate tissue was strongest in comparison to the other newly-identified kallikrein proteins (hK5, hK7, hK8 and hK11). Therefore, KLK4/hK4 expression was characterised further to define this cellular localisation and examined in non-prostatic tissue and also in a larger number of prostate tissues in an attempt to determine its potential value as a biomarker for prostate disease. Three hK4 antipeptide polyclonal antibodies, derived against N-terminal, mid-region and C-terminal hK4 amino acid sequences, were used. The hK4 N-terminal antipeptide antibody was used to demonstrate the cellular localisation of hK4 in kidney, salivary glands, liver, testis, colon carcinoma, heart, endometrium and ovarian cancer, for the first time. The presence of hK4 in these non-prostate tissues was consistent with the previous reports using RT-PCR. The dual cytoplasmic and nuclear localisation of hK4 observed in the prostate above was also seen in these tissues. Although hK4 was found widely expressed in many human tissue types, indicating that it is not prostate specific in its expression, the highest expression level of hK4 was seen in the prostate. Therefore, detailed expression patterns and levels of KLK4 mRNA and hK4 protein in the normal prostate and prostatic diseases and histopathological lesions were investigated and reported for the first time in this study. Twelve benign prostatic hyperplasia (BPH), 19 adenocarcinoma (Gleason grade 2-5) and 34 bone metastases from prostate cancer were analysed. Using in situ hybridisation, the expression of KLK4 mRNA was detected in the cytoplasm of the secretory cells of both normal and diseased prostate tissue. KLK4 mRNA was also noted in both secretory and basal cells of PIN lesions, but the basal cells of normal glands were negative. Using the hK4 N-terminal and mid-region antipeptide antibodies, hK4 was predominantly localised in the cytoplasm of the secretory cells. The intensity of hK4 staining appeared lowest in normal and BPH, and increased in PIN lesions, high Gleason grade prostate cancer and bone metastases indicating the potential of hK4 as a histopathological marker for prostatic neoplasias. Further studies are required with a larger cohort to determine its utility as a clinical biomarker. Small foci of atypical cells, which were found within normal glands, were also intensely stained. Surprisingly, hK4 protein was found in the nucleus of the secretory cells (but not the basal cells) of high grade PIN and Gleason grade 3 prostate cancer. The detection of KLK4 mRNA and hK4 protein in PIN lesions and small foci of atypical cells suggests that up-regulation of KLK4 expression occurs early in the pathology of prostate carcinogenesis. The finding of basal cell expression is not typical for the kallikreins and it is not clear what role hK4 would play in this cell type. With the use of the hK4 C-terminal antipeptide antibody, the staining was mainly localised in the nuclei of the secretory cells of the prostate glands. Although the nuclear localisation was readily noted in more than 90% of epithelial cells of the prostate gland with the C-terminal antibody, no difference in staining intensity was observed among the histopathological lesions of the prostate. The prominent nuclear localisation with the C-terminal antipeptide antibody was also shown to be distributed throughout the nucleus by using confocal microscopy. Further, by using gold-labelled particles for electron microscopy, the intracellular localisation of these hK4 antipeptide antibodies was reported here for the first time. Similar to the immunohistochemical results, the cytoplasm was the major site of localisation with the N-terminal and mid-region antipeptide antibodies. To further characterise the involvement of KLK4/hK4 in human prostate cancer progression, the transgenic adenocarcinoma mouse prostate (TRAMP) model was used in this study. In this study, mouse KLK4 (also known as enamel matrix serine protease -1, EMSP-1) was shown to be expressed in the TRAMP prostate for the first time. Previous studies had only shown the developing tooth as a site of expression for EMSP-1. The level of EMSP-1 mRNA expression was increased in PIN and prostate cancer lesions of the TRAMP model, while negative or low levels of EMSP-1 mRNA were seen in normal glands or in control mouse prostate tissue. The normal mouse prostate did not stain with any the three hK4 antipeptide antibodies. hK4 N-terminal and mid-region antipeptide antibodies showed positive staining in the cytoplasm of the epithelial cells of PIN and cancer lesions of the mouse prostate. The C-terminal antipeptide antibody showed distinctively nuclear staining and was predominantly localised in the nuclei of the glandular cells of PIN and cancer lesions of the mouse prostate. The expression patterns of both the mRNA and protein level for mouse KLK4 strongly supported the observations of KLK4/hK4 expression in the human prostate and further support the utility of the TRAMP model. Overall, the findings in this thesis indicate a clear association of KLK4/hK4 expression with prostate cancer progression. In addition, several intriguing findings were made in terms of cellular localisation (basal as well as secretory cells; nuclear and cytoplasmic) and high expression in atypical glandular cells and PIN, perhaps indicating an early involvement in prostate disease progression and, additionally, utility as basal cell and PIN histological markers. These findings provide the basis for future studies to confirm the utility of hK4 as a biomarker for prostate cancer progression and identify functional roles in the different cellular compartments.