Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) coordinate tissue development and postnatal remodelling by regulating proliferation, differentiation and apoptosis. However, their role in wound healing remains unclear. To study this, transgenic mice overexpressing Smad1 (K14-caSmad1) or the BMP antagonist Noggin (K14-Noggin) were utilised, together with human and mouse ex vivo wound healing models and in vitro keratinocyte culture. In wild-type mice, transcripts for Bmpr-1A, Bmpr-II, Bmp ligands and Smad proteins were decreased following tissue injury, whilst Bmpr-1B expression was up-regulated. Furthermore, immunohistochemistry revealed a down-regulation of BMPR-1A in hair follicles adjacent to the wound in murine skin, whilst in murine and human wounds, BMPR-1B and phospho-Smad-1/5/8 expression was pronounced in the wound epithelial tongue. K14-caSmad1 mice displayed retarded wound healing, associated with reduced keratinocyte proliferation and increased apoptosis, whilst K14-Noggin mice exhibited accelerated wound healing. Furthermore, microarray analysis of K14-caSmad1 epidermis revealed decreased expression of distinct cytoskeletal and cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a versus controls. Human and mouse keratinocyte proliferation and migration were suppressed by BMP-4/7 both in vitro and ex vivo, whilst they were stimulated by Noggin. Additionally, K14-caSmad1 keratinocytes showed retarded migration compared to controls when studied in vitro. Furthermore, Bmpr-1B silencing accelerated migration and was associated with increased expression of Krt16, Krt17 and Myo5a versus controls. Thus, this study demonstrates that BMPs inhibit proliferation, migration and cytoskeletal re-organization in epidermal keratinocytes during wound healing, and raises a possibility that BMP antagonists may be used for the future management of chronic wounds.

Antagonism of the activin receptor signaling pathway represents a promising potential therapy for the muscular dystrophies and other muscle wasting disorders (i.e., cachexia or sarcopenia). Previous research has shown that antagonism of activin signaling promotes muscle growth, attenuates muscle wasting, and restores function in both wild type and diseased animals. Our laboratory has recently developed a novel small molecule (SGI-1252) that inhibits activin downstream (i.e., Smad2/3 phosphorylation) signaling. Purpose: In this study we determined how eight weeks of orally administered SGI-1252 affected TGF-ß signaling, whole body mass, individual limb muscle mass, and muscle fiber cross sectional area (CSA). Methods: Wild-type (WT) mice were treated with SGI-1252 or a vehicle control (VC) via oral gavage (400 mg/kg 3 times per week) for 8 weeks. Body mass was measured twice per week during the 8-week treatment period. At the end of the treatment period, gastrocnemius and tibialis anterior (TA) muscles were excised, weighed, and prepared for histological and biochemical analyses. Results: Following 8 weeks of treatment, there was no difference in weight gain between SGI-1252 (24.8 ± 1.8g) and VC treated mice (23.2 ± 1.5g) (p = 0.06). Gastrocnemius whole muscle mass was significantly greater in the SGI-1252 treated group relative to the VC treated mice (139.6 ± 12.8 mg vs 128.8 ± 14.9 mg) (p = 0.04), although when normalized with body mass there was no difference in gastrocnemius mass. For the TA muscle, there were no significant differences in whole muscle mass between SGI-1252 and VC groups, yet TA muscles in the SGI-1252 treated group had a reduced muscle fiber CSA compared to controls (621 ± 44 µm2 vs 749 ± 36 µm2) (p = 0.0005). There was a statistical trend of decreasing Smad2 phosphorylation in the SGI-1252 treated TA muscles (mean SGI-1252 = 0.668 vs VC = 0.848) (p = 0.06), and no significant differences in Smad2 phosphorylation in the gastrocnemius. Conclusions: Contrary to our hypothesis, 8 weeks of orally administered SGI-1252 was not effective in promoting increases in whole body mass, limb whole muscle mass, or myofiber cross sectional area. This may be due to the inability of SGI-1252, at the administered dose, to effectively decrease signaling downstream of the activin receptor. Clearly, studies using a wider range of doses and delivery methods will be needed to ascertain the efficacy of SGI-1252 as a potential therapeutic.

Transforming growth factor β (TGF-β) is a member of a large family of cytokines that regulate many crucial events in cells, including proliferation, differentiation, migration and apoptosis. Deregulated TGF-β signaling is associated with various forms of cancers and developmental disorders. TGF-β binds to a receptor complex at the surface of cells and activates a signaling cascade involving specific intracellular signaling proteins, known as Smads. Following receptor activation, the Smads are activated by phosphorylation and translocate to the nucleus, where they activate or repress the expression of specific genes. Posttranslational modifications regulate the function of proteins in a number of ways, including their activity, stability, localization, and/or interactions with other proteins. These modifications are important to modulate the strength and specificity of cellular signal transduction. Smad7, an important negative modulator of TGF-β signaling, has been shown to be acetylated by the acetyltransferase p300. My aim was to further explore the involvement of protein acetylation in TGF-β-dependent signaling. We could show that the acetylation of Smad7 is a reversible process. Interestingly, earlier work had shown that the acetylation of Smad7 prevented its degradation. In agreement with this observation, we found that the ubiquitylation and degradation of Smad7 was increased following cotransfection with HDAC1, a protein deacetylase. Based on our observations, we propose a model in which the stability of Smad7 is controlled by the balance between its acetylation, deacetylation and ubiquitylation. In a separate study, we found that also Smad2 and Smad3 are acetylated by p300/CBP and P/CAF upon TGF-β stimulation. Moreover, we found that the acetylation of the short isoform of Smad2 promoted its DNA binding activity, resulting in an increased transcriptional activity. Our results suggest that the increased DNA binding in response to acetylation is due to a conformational change in Smad2.

A comprehensive understanding of how the body and all its components function is essential when this knowledge is exploited for medical purposes. The achievements in biological and medical research during last decades has provided us with the complete human genome and identified signaling pathways that governs the cellular processes that facilitates the development and maintenance of higher order organisms. This has brought about the realization that diseases such as cancer is a consequence of genomic aberrations that effects these signaling pathways, endowing cancer cells with the capacity to circumvent homeostasis by acquiring features like self-sustained proliferation and insensitivity to apoptosis. The increased understanding of biology and medicine has been made possible by the development of advanced methods to carry out biological and clinical analyses. The demands of a method often differ regarding in what context it will be applied. It may be acceptable for method to be laborious and time consuming if it is used in basic research, but for medical purposes molecular methods need to be fast and straightforward to perform. Innovative technologies should preferentially address the demands of both researchers and clinicians and provide data not possible to obtain by other methods. An example of such a method is the in situ proximity ligation assay (in situ PLA). In this thesis I have used this method to determine the activity status, at the single-cell level, of the transforming growth factor-β (TGF-β) signaling pathway and activating protein-1 (AP-1) family of transcription factors.  Both of these pathways are frequently involved in cancer development and progression. In addition to this research I herein also present further modifications of in situ PLA, and analyses thereof, to increase the utility and resolution of this assay.

Οι σπειραματονεφρίτιδες (ΣΝ) αποτελούν μια ομάδα νοσημάτων του σπειράματος ανοσολογικής αιτιολογίας που σε πολλές περιπτώσεις οδηγούν στην πλήρη καταστροφή του νεφρικού παρεγχύματος με αποτέλεσμα τη νεφρική ίνωση και την ανάγκη υποκατάστασης της νεφρικής λειτουργίας με μεθόδους εξωνεφικής κάθαρσης. Τα τελευταία χρόνια έχει σημειωθεί σημαντική πρόοδος στην ανίχνευση των μοριακών μηχανισμών που οδηγούν στη νεφρική ίνωση. Κεντρικό ρόλο στη διαδικασία αυτή φαίνεται να διαδραματίζει το Smad σηματοδοτικό μονοπάτι που διαμεσολαβεί τα σήματα του TGF-β στα νεφρικά κύτταρα. Ο ρόλος του TGF-β στη νεφρική βλάβη είναι πλέον καλά τεκμηριωμένος. Αντίθετα ελάχιστα είναι γνωστά για την έκφραση και τη λειτουργία του Smad σηματοδοτικού μονοπατιού στις ανθρώπινες ΣΝ. Ο σκοπός της παρούσας μελέτης ήταν η διερεύνηση της έκφρασης των παραγόντων pSmad2/3, p300, Sp1, Smad7 και Ski (μορίων που συμμετέχουν στο Smad σηματοδοτικό μονοπάτι) σε νεφρικές βιοψίες ασθενών με ΣΝ και η ανίχνευση της δράσης τους όσον αφορά την παθογένεση και την εξέλιξη της νεφρικής βλάβης. Χρησιμοποιήθηκαν 157 βιοψίες ασθενών με ΣΝ και 15 φυσιολογικά νεφρικά δείγματα από ασθενείς που είχαν υποβληθεί σε νεφρεκτομή για καρκίνο του νεφρού. Οι σπειραματονεφρίτιδες κατηγοριοποιήθηκαν σε πρωτοπαθείς (n=91) και δευτεροπαθείς (n=66) ή υπερπλαστικές (n=86) και μη υπερπλαστικές (n=71). Η μελέτη της έκφρασης των pSmad2/3, p300, Sp1, Smad7 και Ski έγινε με τη χρήση ανοσοϊστοχημικής μεθόδου. Έγιναν συσχετίσεις με κλινικά δεδομένα των ασθενών (κρεατινίνη ορού και λεύκωμα ούρων 24ώρου), καθώς και δείκτες ιστολογικής βλάβης (σπειραματοσκλήρυνση, διάμεση ίνωση, σωληναριακή ατροφία και διάμεση φλεγμονή). Παρατηρήθηκε πολύ μεγάλη αύξηση της έκφρασης των pSmad2/3, Sp1 και p300 σε όλα τα σπειραματικά κύτταρα των ΣΝ σε σχέση με την ομάδα ελέγχου. Επίσης, αυξήθηκε η έκφραση του πυρηνικού Smad7 στις υπερπλαστικές κυρίως ΣΝ με ταυτόχρονη εξάλειψη της κυτταροπλασματικής έκφρασής του σε όλες τις ΣΝ και παράλληλη μείωση της έκφρασης του Ski. Όσον αφορά τα σωληνάρια, στα εγγύς παρατηρήθηκε αύξηση της έκφρασης των pSmad2/3, Sp1 και p300 σε όλες τις ΣΝ και αύξηση του πυρηνικού Smad7 στις υπερπλαστικές, ενώ στα άπω και τα αθροιστικά παρατηρήθηκε αύξηση της έκφρασης του pSmad2/3 και του πυρηνικού Smad7 με ταυτόχρονη μείωση του κυτταροπλασματικού Smad7. Το Ski παρουσίασε σημαντική μείωση της έκφρασής του στις ΣΝ σε όλους τους τύπους σωληναρίων. Η σπειραματική έκφραση των pSmad2/3, p300 και του πυρηνικού Smad7 ήταν αυξημένη στις υπερπλαστικές ΣΝ και κυρίως τις δευτεροπαθείς. Η σωληναριακή έκφραση των pSmad2/3 και Smad7 και η εγγύς σωληναριακή των p300, Sp1 και του κυτταροπλασματικού Smad7 ήταν επίσης αυξημένες στις υπερπλαστικές ΣΝ. Αντίθετα, το Ski δεν παρουσίασε διαφορές στην έκφραση μεταξύ των υπερπλαστικών και μη υπερπλαστικών ΣΝ. Ακόμη, ανιχνεύθηκαν σημαντικές συσχετίσεις μεταξύ μεταξύ της έκφρσης των pSmad2/3, p300, Sp1 και Smad7 στο σπείραμα και τα εγγύς σωληνάρια. Η σπειραματική και εγγύς σωληναριακή έκφραση των pSmad2/3, Sp1 και η σπειραματική του πυρηνικού Smad7 έδειξαν να συσχετίζονται θετικά με τα επίπεδα της κρεατινίνης του ορού, ενώ η έκφραση αυτών και του p300 σε διαφόρους τύπους σωληναρίων εμφάνισαν θετική στσχέτιση με δείκτες ιστολογικής βλάβης. Τέλος η έκφραση όλων των μορίων (εκτός του Ski) ήταν συχνότατη στις στοιχειώδεις σπειραματικές βλάβες. Συμπερασματικά, το Smad σηματοδοτικό μονοπάτι φαίνεται να ενεργοποιείται στις ανθρώπινες ΣΝ. Μάλιστα, η ενεργοποίησή του αυτή πιθανώς συμβάλλει στην παθογένεση αλλά και την εξέλιξη της νεφρικής βλάβης. Μελλοντικές θεραπευτικές στρατηγικές που θα στοχεύουν στην αναστολή του μονοπατιού αυτού ίσως συμβάλλουν στην παρεμπόδιση της εξέλιξης και τη θεραπεία των ανθρωπίνων ΣΝ. / -

The transforming growth factor beta (TGFβ)/activin signaling pathway has a number of documented roles during wound healing and is becoming increasingly appreciated as a vital component of multi-tissue regeneration. The leopard gecko (Eublepharis macularius) is able to spontaneously, and repeatedly, regenerate its tail following tail loss. We thus examined the expression and localization of several key components of the TGFβ/activin signaling pathway during tail regeneration of the leopard gecko. We observed a marked increase in phosphorylated-Smad2 expression among regenerating tissues corresponding to the location of the regenerate blastema. Interestingly, we observe that during early regeneration there appears to be an absence of TGFβ family member TGFβ1 and instead a strong upregulation of activin-βA. We also observe the expression of EMT transcription factors Snail1 and Snail2 in blastemal tissue. These observations combined with other data provide strong support for the importance of unique and non-overlapping expression patterns of different TGFβ ligands during multi-tissue regeneration

<p>The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and homeostasis. BMPs have been shown to pattern the dorsoventral axis in zebrafish (<italic>Danio rerio</italic>) early during embryonic development by establishing a dorsal-to-­ventral ligand gradient. During tumorigenesis, BMPs primarily function as tumor promoters, as an increase in BMP expression is associated with an increase in invasion, migration, epithelial-­to-­mesenchymal transition (EMT), proliferation and angiogenesis.</p><p>Although it is clear that BMPs play multiple roles in these biological events, the precise mechanism by which BMPs mediate these functions is not fully understood. Canonically, BMP ligands signal through cell surface receptor complexes that phosphorylate transcription factors, Smad1, Smad5 and Smad8, which mediate BMP-­ specific gene transcription. While studying BMP signaling during cancer progression, we determined that BMPs unexpectedly signal through the canonical TGF-&beta;-­responsive transcription factors, Smad2 and Smad3.</p><p>We determined that BMP-­induced Smad2/3 signaling occurs preferentially in embryonic cells and transformed cells. BMPs signal to Smad2/3 by stimulating complex formation between the BMP binding TGF-­&beta; superfamily receptors, ALK3/6, and the Smad2/3 phosphorylating receptors, ALK5/7. BMP signaling through Smad1/5/8 induces MET, while Smad1/5 and Smad2 mediate dorsoventral axis patterning in zebrafish embryos and Smad3 facilitates invasion.</p><p>Taken together, our data provides evidence that BMP-­induced Smad2 and Smad3 phosphorylation occurs through a non-­canonical signaling mechanism to mediate multiple biological events. Thus, the signaling mechanisms utilized by BMPs and TGF-­&beta; superfamily receptors are broader than previously appreciated.</p> / Dissertation

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Biochemistry." Includes bibliographical references (p. 191-203).

Thesis (Ph. D.)--Cornell University, May, 2009. / Vita. Includes bibliographical references (leaves 152-178).

Human pluripotent stem cells (hPSCs) are an invaluable model for cellular and developmental biology, and hold great potential for translational applications. While great progress has been made in elucidating the signalling pathways regulating pluripotency and differentiation, our mechanistic understanding of the downstream regulations is still incomplete. Moreover, studies aimed at clarifying these aspects are severely impeded by the lack of efficient methods to conditionally modulate gene expression in hPSCs and hPSC-derived cells. In this dissertation I provide new insights into the molecular mechanisms controlled by the Activin/Nodal-SMAD2/3 signalling pathway, whose activity dictates the balance between hPSC pluripotency and differentiation. First, I show that SMAD2/3 modulates the chromatin epigenetic landscape of hPSCs by cooperating with the pluripotency factor NANOG to recruit the DPY30-COMPASS complex and promote histone 3 lysine 4 trimethylation (H3K4me3). This regulation promotes expression of pluripotency genes, while poising developmental regulators for activation during differentiation. Secondly, I describe a novel efficient approach for inducible gene knockdown in hPSCs and hPSC-derived cells. By taking advantage of this technology, I demonstrate that DPY30 is required for early differentiation of hPSCs into certain mesoderm and endoderm derivatives. Finally, I report the first large-scale proteomic identification of SMAD2/3 interacting proteins in both undifferentiated and differentiating hPSCs. This analysis not only confirms that SMAD2/3 interacts with multiple epigenetic modifiers involved in hPSC fate choices, but also implicates SMAD2/3 in several functions other than transcriptional regulation. In particular, I describe how SMAD2/3 physically and functionally interacts with the METTL3-METTL14-WTAP complex to promote the formation of N6-methyladenosine (m6A). This epitranscriptional modification antagonizes the expression of selected mRNAs, including pluripotency factors whose transcription is promoted by SMAD2/3. Therefore, this provides a negative feedback that facilitates rapid exit from pluripotency upon inhibition of Activin/Nodal signalling. Overall, the work presented in this dissertation advances the stem cell field in two ways. First, it demonstrates that the Activin/Nodal-SMAD2/3 pathway finely orchestrates the balance between pluripotency and differentiation by shaping both the epigenome and the epitranscriptome of hPSCs. Secondly, it provides a novel powerful technology to facilitate further studies of the mechanisms that regulate cell fate decisions.