Generation and Characterization of Knockout Mice and Analysis of Patient Material Demonstrates a Role for Tissue Inhibitor of Metalloproteinases 4 (Timp4) in the Cardiovascular System and Tumor Metastasis

This thesis focuses on tissue inhibitor of metalloproteinases 4 (TIMP4) which is the newest member of a small gene and protein family of four closely related endogenous inhibitors of extracellular matrix (ECM) degrading enzymes. Existing data on TIMP4 suggested that it exhibits a more restricted expression pattern than the other TIMPs with high expression levels in heart, brain, ovary and skeletal muscle. These observations and the fact that the ECM is of special importance to provide the cardiovascular system with structural strength combined with elasticity and distensibility, prompted the present molecular biologic investigation on TIMP4. In the first part of the study the murine Timp4 gene was cloned and characterized in detail. The structure of murine Timp4 genomic locus resembles that in other species and of the other Timps. The highest Timp4 expression was detected in heart, ovary and brain. As the expression pattern of Timp4 gives only limited information about its role in physiology and pathology, Timp4 knockout mice were generated next. The analysis of Timp4 knockout mice revealed that Timp4 deficiency has no obvious effect on the development, growth or fertility of mice. Therefore, Timp4 deficient mice were challenged using available cardiovascular models, i.e. experimental cardiac pressure overload and myocardial infarction. In the former model, Timp4 deficiency was found to be compensated by Timp2 overexpression, whereas in the myocardial infarct model, Timp4 deficiency resulted in increased mortality due to increased susceptibility for cardiac rupture. In the wound healing model, Timp4 deficiency was shown to result in transient retardation of re-epithelialization of cutaneous wounds. Melanoma tumor growth was similar in Timp4 deficient and control mice. Despite of this, lung metastasis of melanoma cells was significantly increased in Timp4 null mice. In an attempt to translate the current findings to patient material, TIMP4 expression was studied in human specimens representing different inflammatory cardiovascular pathologies, i.e. giant cell arteritis, atherosclerotic coronary arteries and heart allografts exhibiting signs of chronic rejection. The results showed that cardiovascular expression of TIMP4 is elevated particularly in areas exhibiting inflammation. The results of the present studies suggest that TIMP4 has a special role in the regulation of tissue repair processes in the heart, and also in healing wounds and metastases. Furthermore, evidence is provided suggesting the usefulness of TIMP4 as a novel systemic marker for vascular inflammation.

The Effects of Fibroblast Growth Factor 8b on Reproductive Organs and Prostate Tumorigenesis

Fibroblast growth factors (FGFs) are involved in the development and homeostasis of the prostate and other reproductive organs. FGF signaling is altered in prostate cancer. Fibroblast growth factor 8 (FGF8) is a mitogenic growth factor and its expression is elevated in prostate cancer and in premalignant prostatic intraepithelial neoplasia (PIN) lesions. FGF8b is the most transforming isoform of FGF8. Experimental models show that FGF8b promotes several phases of prostate tumorigenesis - including cancer initiation, tumor growth, angiogenesis, invasion and development of bone metastasis. The mechanisms activated by FGF8b in the prostate are unclear. In the present study, to examine the tumorigenic effects of FGF8b on the prostate and other FGF8b expressing organs, an FGF8b transgenic (TG) mouse model was generated. The effect of estrogen receptor beta (ERβ) deficiency on FGF8binduced prostate tumorigenesis was studied by breeding FGF8b-TG mice with ERβ knockout mice (BERKOFVB). Overexpression of FGF8b caused progressive histological and morphological changes in the prostate, epididymis and testis of FGF8b-TG-mice. In the prostate, hyperplastic, preneoplastic and neoplastic changes, including mouse PIN (mPIN) lesions, adenocarcinomas, sarcomas and carcinosarcomas were present in the epithelium and stroma. In the epididymis, a highly cancer-resistant tissue, the epithelium contained dysplasias and the stroma had neoplasias and hyperplasias with atypical cells. Besides similar histological changes in the prostate and epididymis, overexpression of FGF8b induced similar changes in the expression of genes such as osteopontin (Spp1), connective tissue growth factor (Ctgf) and FGF receptors (Fgfrs) in these two tissues. In the testes of the FGF8b-TG mice, the seminiferous epithelium was frequently degenerative and the number of spermatids was decreased. A portion of the FGF8b-TG male mice was infertile. Deficiency of ERβ did not accelerate prostate tumorigenesis in the FGF8b-TG mice, but increased significantly the frequency of mucinous metaplasia and slightly the frequency of inflammation in the prostate. This suggests putative differentiation promoting and anti-inflammatory roles for ERβ. In summary, these results underscore the importance of FGF signaling in male reproductive organs and provide novel evidence for a role of FGF8b in stromal activation and prostate tumorigenesis.

JNK regulation of neurogenesis, neuroplasticity and anxiety-related behaviors in mice

I vår moderna värld har förekomsten av ångeststörningar drastiskt ökat, vilket påverkar välfärden i våra samhällen. Eftersom de molekylära mekanismerna bakom ångest är relativt okända, är möjligheterna till behandling av ångeststörningar begränsade. I och med utvecklingen av genetiska manipuleringmetoder och avbildningstekniker har strukturella förändringar associerade med ångeststörningar kunnat konstateras. Neuroanatomiska studier har påvisat störningar i dendritarkitektur, dendrittaggar och i neurogenesen hos vuxna individer. Särskilt neurogenesen i hippocampus anses viktig i detta sammanhang. Neurogenes i hippocampus har föreslagits spela en viktig roll i ångeststörningarnas patofysiologi och för hur vissa antidepressiva läkemedel förmedlar sin effekt. Under senare år har MAP-kinaser (MAPK) föreslagits vara involverade både i uppkomsten av affektiva störningar och i neurogenes i hippocampus. JNK är en grupp kinaser inom MAPK-familjen som aktiveras av olika externa stressfaktorer. I normala celler är aktiviteten hos JNK låg. Cell-stress ökar aktiviteten hos JNK vilket leder till bl.a. apoptos. JNK kinaser anses vara potentiella terapeutiska mål för behandling av neurodegenerativa sjukdomar men deras potential som mål för behandling av affektiva sjukdomar har tillsvidare inte utretts. Den här avhandlingen behandlar betydelsen av JNK för ångestrelaterat beteende hos möss. Med hjälp av Jnk1-knockout möss och farmakologisk inhibering av JNK-signalering, demonstreras att JNK reglerar neurogenes i hippocampus, vilket i sin tur ligger bakom mössens ångestrelaterade beteende. Jnk1-knockout möss var mindre ängsliga och uppvisade ökad neurogenes i hippcampus jämfört med kontrollmöss. Inhibering av JNK-signalering i hjärnan hos möss gjorde dem också mindre ängsliga och ökade neurogenesen i hippocampus på samma sätt som vissa antidepressiva läkemedel. Inhibering av JNK-aktivitet ledde inte bara till ökad neurogenes i hippocampus, utan främjade också mognandet av nybildade nervceller hos vuxna möss. Resultaten visar också att dendritarkitekturen och fördelningen av dendrittaggar hos CA3 neuroner i hippocampus är förändrad hos Jnk1-knockout möss. Genom screening av substratmolekyler för JNK hittades två JNKeffektormolekyler, MARCKSL1 (ett aktin-associerat protein) och MAP2 (ett mikrotubulus-associerat protein), som reglerade neuronernas sttruktur. Det här tyder på att JNK-signalering kan kontrollera ångeststörningsrelaterade förändringar hor dendriter och dendrittaggar. Sammanfattningsvis ger resultaten som presenteras i avhandlingen en ökad insikt i molekylära mekanismer som kan leda till ångeststörningsrelaterade förändringar i neurogenes och dendritstruktur. Därtill föreslås att JNKsignalbanan har potential som terapeutiskt mål för behandling av ångeststörningar.