Downregulation of lung mitochondrial prohibitin in COPD

Summary Prohibitins (PHB1 and PHB2) are versatile proteins located at the inner mitochondrial membrane, maintaining normal mitochondrial function and morphology. They interact with the NADH dehydrogenase protein complex, which is essential for oxidoreductase activity within cells. However, their expression in lung epithelium, especially in smokers and patients with inflammatory lung diseases associated with increased oxidative stress, such as COPD, is unknown. Lung tissue specimens from 45 male subjects were studied: 20 COPD patients [age: 65.7 AE 5.8 years, smoking: 84.6 AE 33.6 pack-years, FEV 1 (%pred.): 58.7 AE 14.6, FEV 1 /FVC (%): 63.8 AE 9.4], 15 non-COPD smokers [age: 59.0 AE 12.1 years, smoking: 52.5 AE 20.8 pack-years, FEV 1 (%pred.): 85.5 AE 14.2, FEV 1 /FVC (%): 78.5 AE 4.7] and 10 non-smokers. Quantitative real-time PCR experiments were carried out for PHB1 and PHB2, using b-actin as internal control. Non-COPD smokers exhibited lower PHB1 mRNA levels when compared to non-smokers (0.55 AE 0.06 vs. 0.90 AE 0.06, P Z 0.043), while PHB1 expression was even further decreased in COPD patients (0.32 AE 0.02), a statistically significant finding vs. both non-COPD smokers (P Z 0.040) and non-smokers (P < 0.001). By contrast, PHB2 levels were similar among the three study groups. Western blot analysis for the PHB1 protein verified the qPCR results (non-smokers: 1.77 AE 0.13; non-COPD smokers: 0.97 AE 0.08; COPD patients: 0.59 AE 0.10, P Z 0.007). Further analysis revealed that PHB1 downregulation in COPD patients cannot be attributed solely to smoking, and that PHB1 expression levels are associated with the degree of airway obstruction [FEV 1 (P mRNA Z 0.004, P protein Z 0.014)]. The significant downregulation of PHB1 in COPD and non-COPD smokers in comparison to non-smokers possibly reflects a distorted mitochondrial function due to decreased mitochondrial stability, especially in the mitochondria of COPD patients.

Μελέτη των επιπέδων έκφρασης των πεπτιδικών αυξητικών παραγόντων και των γονιδίων P53, CYCLIN D1 και TPL-2/COT στην υπερπλασία και τον καρκίνο του προστάτη

The prostate grand is retroperitoneal organ that surrounds the bladder neck and the urethra. Its function is the secretion of the prostatic fluid, which is rich in electrolytes and enzymes, in order to provide a source of energy for the spermatozoa and to increase their mobility. Benign prostatic hyperplasia is the most common benign tumor among men aged above 60. The suggested mechanisms that lead to the development of the disease are the co-operativity between estrogens and androgens and the disruption of the balance between the rate of proliferation and apoptosis of the prostate cells, which is caused by hormonal and developmental factors, as well as by oncogenes. Prostate cancer is the most frequent cancer among men and the second leading cause of death behind lung cancer. Its characteristics are molecular and genetic disorders that cause rapid cellular proliferation and inhibit apoptosis. Genetic background, hormonal disorders, exposure in environmental carcinogens and sexually transmitted diseases contribute significantly to the development of the disease. The subject of this thesis was to determine the role of peptide growth factors and the genes p53, Cyclin D1 και Tpl-2/Cot in the development of both benign and malignant prostatic diseases. For this cause we collected tissue samples from 40 patients with prostate adenocarcinoma, from 40 patients with benign prostatic hyperplasia and from 10 normal donors (collected post-mortem). In the first part of the thesis we studied the expression of peptide growth factors in normal, benign and malignant prostate. Peptide growth factors are regulatory proteins that participate in cellular growth, differentiation and apoptosis. Several oncogenes are in fact growth factors or growth factor receptors. The growth factors studied were: FGF2 (induces proliferation of epithelial and stromal cells and is a powerful angiogenic factor), EGF (its role is important in embryogenesis, in cellular differentiation and in angiogenesis), TGFΒ1 (induces angiogenesis and the formation of the extracellular matrix, represses the immune system and regulates cellular differentiation), VEGF (is involved in the formation of new blood vessels during embryonic development and various other normal and malignant conditions, forming vessels with increased permeability) και IGF1 (induces the proliferation of stromal and epithelial cells). Peptide growth factors are important in normal prostatic development and differentiation, while their presence has been confirmed in prostate cell lines and tumors. Our results showed that growth factors VEGF, EGF and FGF2 are overexpressed in prostate cancer, while growth factors TGFB1 and IGF1 have reduced expression. In prostatic hyperplasia the expression of growth factors FGF2 and EGF is normal, while mRNA downregulation was observed in growth factors VEGF, TGFB1 and IGF1. Statistical analysis revealed that prostate cancer patients with high PSA blood levels downregulate FGF2 (p=0.016). Additionally, cancer patients with low Gleason score (50% of human malignancies. p53 has a common polymorphism in codon 72 (Pro to Arg), which alters the biochemical properties of the protein. Our results showed that p53 is overexpressed in prostate cancer, while its levels are downregulated in prostatic hyperplasia. Moreover, the Arg72Pro polymorphism is associated with the development of benign prostatic hyperplasia (p=0.042), but not of prostate cancer. However, patients with prostate cancer that carry the Arg/Arg codon 72 genotype have higher p53 mRNA levels than patients with the other two polymorphic genotypes. Cyclin D1 is a member of the G1 cyclin family, that regulate cell cycle in G1 phase through the molecular pathway of Rb. CCND1 mRNA is alternatively spliced, a procedure that is regulated by a polymorphism (G870A) in a conserved region of exon 4. Our study showed that cyclin D1 is overexpressed in prostate cancer, while it is downregulated in benign prostatic hyperplasia. G870A polymorphism is associated with the development of prostate carcinogenesis (p=0.015), and with early disease onset (p=0.049), but not with the development of prostatic hyperplasia. On the contrary, cyclin D1 expression levels are not affected by the status of the G87A polymorphism. From the above results it is obvious that both p53 and cyclin D1, and their polymorphisms, are implicated in the development of both prostatic disorders. In the second part of the thesis we examined the expression of Tpl-2/Cot kinase. Tpl-2/Cot protooncogene encodes an serine-threonine kinase that is a member of the ΜΑΡΚ family. It activates Erl1/Erk2, JNK and p38MAPK kinases in various cell lines and induces apoptosis through caspases 3 and 9. It also increases the expression of interleukin-2 and TNF-a, activation nuclear transcription factors NFAT and NFkB. Tpl-2/Cot is expressed in most normal tissues in low levels, while it is overexpressed in various malignancies. According to our results, Tpl-2 mRNA were higher in 50% of cancer samples, while its expression was deregulated in benign hyperplasias, since it was overexpressed in 18% of samples and downregulated in 37%. Statistical analysis revealed that Tpl-2 overexpression is observed in prostate cancer patients with age 50% των ανθρώπινων νεοπλασιών. Το p53 έχει έναν πολυμορφισμό στο κωδικόνιο 72 (Pro σε Arg), ο οποίος αλλάζει τις βιοχημικές ιδιότητες της πρωτεΐνης. Τα αποτελέσματα των αναλύσεων μας έδειξαν ότι το p53 υπερεκφράζεται στον καρκίνο του προστάτη, ενώ τα επίπεδα του υποεκφράζονται στην προστατική υπερπλασία. Επιπρόσθετα, ο πολυμορφισμός Arg72Pro σχετίζεται με την εμφάνιση προστατικής υπερπλασίας (p=0.042) αλλά όχι με την εμφάνιση καρκίνου. Ωστόσο οι ασθενείς με καρκίνο του προστάτη που φέρουν το γονότυπο Arg/Arg εμφανίζουν αυξημένα επίπεδα έκφρασης του p53, σε σχέση με τους φορείς των υπολοίπων γονοτύπων. Η Cyclin D1 είναι μέλος της οικογένειας των G1 κυκλινών, οι οποίες ρυθμίζουν τον κυτταρικό κύκλο στη φάση G1 μέσω του μοριακού μονοπατιού της Rb. Το mRNA της υπόκειται εναλλακτικό μάτισμα, μια διαδικασία που ρυθμίζεται από έναν πολυμορφισμό (G870A) σε μια συντηρημένη περιοχή του εξωνίου 4. Η μελέτη μας έδειξε ότι η Cyclin D1 υπερεκφράζεται στον καρκίνο του προστάτη ενώ υποεκφράζεται στην προστατική υπερπλασία. Ο πολυμορφισμός G870A σχετίζεται με την εμφάνιση προστατικού αδενοκαρκινώματος (p=0.015) και μάλιστα σε νεότερη ηλικία (p=0.049), αλλά όχι και υπερπλασίας προστάτη. Αντιθέτως, τα επίπεδα έκφρασης της Cyclin D1 δεν επηρεάζονται από τον πολυμορφισμό αυτό. Από τα παραπάνω αποτελέσματα είναι φανερό πως τόσο η p53, όσο και η Cyclin D1, καθώς και οι πολυμορφισμοί τους, εμπλέκονται και στις δύο ασθένειες του προστάτη αδένα. Στο τρίτο μέρος της διατριβής εξετάσαμε την έκφραση της κινάσης Tpl-2/Cot. Το πρωτο-ογκογονίδιο Tpl-2/Cot κωδικοποιεί μια πρωτεϊνική κινάση σερίνης-θρεονίνης, η οποία ανήκει στις ΜΑΡΚ κινάσες. Ενεργοποιεί τις κινάσες Erl1/Erk2, JNK και p38MAPK σε διάφορες κυτταρικές σειρές και προάγει την απόπτωση μέσω των κασπασών 3 και 9. Επιπλέον, αυξάνει την έκφραση της ιντερλευκίνης 2 και του TNF-a, ενεργοποιώντας τους παράγοντες μεταγραφής NFAT και NFkB. Εκφράζεται στους περισσότερους φυσιολογικούς ιστούς σε χαμηλά επίπεδα, ενώ εμφανίζει υπερέκφραση σε διάφορους καρκίνους. Σύμφωνα με τα αποτελέσματα των αναλύσεών μας, τα επίπεδα της έκφρασης του Tpl-2 ήταν υψηλότερα από τα φυσιολογικά στο 50% των καρκινικών δειγμάτων, ενώ αντιθέτως η έκφραση του Tpl-2 ήταν απορυθμισμένη στις καλοήθεις υπερπλασίες, αφού είχαμε υπερέκφρασή του στο 18% των δειγμάτων και υποέκφρασή του στο 37%. Η στατιστική ανάλυση αποκάλυψε ότι η υπερέκφραση του παρατηρήθηκε σε ασθενείς με καρκίνο του προστάτη κάτω των 70 ετών (p=0.019), ενώ στην καλοήθη υπερπλασία οι ασθενείς που εμφανίζουν υποέκφραση του Tpl-2 είναι κατά μέσο όρο 5 έτη μεγαλύτεροι από τους ασθενείς με φυσιολογική έκφραση (p=0.039). Από τα αποτελέσματα αυτά συμπεραίνουμε ότι η αύξηση της έκφρασης του Tpl-2 παίζει σημαντικό ρόλο στον καρκίνο του προστάτη και ότι σχετίζεται με πρώιμη έναρξη της νόσου. Επιπλέον η απορύθμιση της έκφρασης του Tpl-2 στα δείγματα με καλοήθη υπερπλασία μπορεί να αποτελεί μέρος ενός μηχανισμού μέσω του οποίου το προστατικό κύτταρο οδηγείται στην υπερπλασία. Τα αποτελέσματα της παρούσης διατριβής παρέχουν σοβαρές ενδείξεις για τη συμμετοχή των μελετηθέντων μορίων στην εμφάνιση της καλοήθους υπερπλασίας και του καρκίνου του προστάτη, ενώ θα μπορούσαν να αξιοποιηθούν για την έγκαιρη διάγνωση, σωστή παρακολούθηση αλλά και θεραπεία των ασθενών

Downregulation of notch signaling pathway in late preterm and term placentas from pregnancies complicated by preeclampsia.

Preeclampsia (PE) is a major cause of maternal mortality and morbidity, affecting 3-5% of all pregnancies. The Notch signaling pathway plays an important role during placental development, activating several target genes. Defects in the Notch pathway have adverse effect on placentation. The aim of this study was to investigate the expression of receptors NOTCH1,-2,-3,-4, ligands DLL1,-3,-4, JAG1,-2 and target genes HEY1,-2 in placental tissue samples from 20 late preterm or term pregnancies complicated by PE versus 20 normal pregnancies. mRNA levels of the studied molecules were measured by quantitative Real-Time PCR (qRT-PCR), while the protein expression of the intracellular domain of NOTCH2 (NICD2) and NOTCH3 (NICD3) was measured by Western Blot (WB). qRT-PCR analysis revealed that NOTCH1, NOTCH4 and DLL1 were not expressed in the placenta. On the contrary, NOTCH2, NOTCH3, DLL3, DLL4, JAG1, JAG2, HEY1 and HEY2 mRNA levels were downregulated in PE samples vs. controls (p<0.01). WB confirmed that NICD2 (p = 0.014) and NICD3 (p<0.001) protein levels were also lower in PE specimens. Statistical analysis revealed several significant associations: of NOTCH3 mRNA expression with smoking during pregnancy (p = 0.029), of NICD3 protein levels (p = 0.028) and DLL3 mRNA levels (p = 0.041) with birth weight centile, and of HEY2 transcript levels with parity (p = 0.034) and mode of delivery (p = 0.028). Our results suggest that Notch pathway downregulation is associated with PE. Further studies are required in order to determine the role of these molecules in PE pathogenesis and to evaluate their potential use for the early detection and treatment of PE

Notch signaling pathway pair-wise mRNA co-expression analysis in normal placentas.

<p><b>CC</b>: Correlation coefficient.</p><p>Notch signaling pathway pair-wise mRNA co-expression analysis in normal placentas.</p

Expression analysis of Notch signaling pathway between normal and PE placentas.

<p><b>P-value (c):</b> crude p-value; <b>P-value (a):</b> P-value adjusted for gestation period, mode of delivery and smoking during pregnancy. All values are presented as mean ± SEM.</p><p>Expression analysis of Notch signaling pathway between normal and PE placentas.</p

Primer sequences, annealing temperatures and amplicon sizes for the qRT-PCR analysis of the studied genes.

<p>Primer sequences, annealing temperatures and amplicon sizes for the qRT-PCR analysis of the studied genes.</p

Clinical characteristics of the study groups.

<p>^a Student’s T test</p><p>^b Mann-Whitney U test</p><p>^c Chi-square test</p><p>^d Fisher’s exact test</p><p>All tests were 2-tailed.</p><p>Clinical characteristics of the study groups.</p