A major limiting factor for long-term, deep-space missions is the radiation dose to astronauts. Because the dose to the astronauts is a mixed field of low- and high-LET radiation, there is a need to understand the effects of both radiation types on whole tissue; however, there are limited published data on the effects of high-LET (linearenergy- transfer) radiation on tissue. Thus, we designed a perfusion chamber system for rat trachea in order to mimic in vivo respiratory tissue. We successfully maintained the perfused tracheal tissue ex vivo in a healthy and viable condition for up to three days. In addition, this project studied the effects of high-LET Fe particles on the overall transformation growth factor (TGF)-beta response after TGF-beta inactivation and compared the results to the TGF-beta response post x-ray irradiation. It was found that a TGF-beta response could be measured in the perfused tracheal tissue, for x-ray and Fe particle irradiations, despite the high autofluorescent background intrinsic to tissue. However, after comparing the TGF-beta response of x-ray irradiation to High-Z-Highenergy (HZE) irradiation, there was not a significant difference in radiation types. The TGF-beta response in x-ray and HZE irradiated perfusion chambers was also measured over time post irradiation. It was found that for 6 hour and 8 hour post irradiation, the TGF-beta response was higher for lower doses of radiation than for higher doses. This is in contrast to the 0 hour fixation which found the TGF-beta response to increase with increased dose. The inverse relationship found for 6 hour and 8 hour fixation times may indicate a threshold response for TGF-beta response; i.e., for low doses, a threshold of dose must be reached for an immediate TGF-beta response, otherwise the tissue responds more slowly to the irradiation damage. This result was unexpected and will require further investigation to determine if the threshold can be determined for the 250 kVp x-rays and 1 Gev Fe particles.

There is an urgent need to define the key pathological driving events in human cutaneous squamous cell carcinoma (cSCC) in order to identify novel therapeutic targets. Already the second most common form of human non-melanoma skin cancer, the incidence of cSCC has continued to rise at epidemic proportions over the past two decades. Rarely, cSCC can be highly aggressive, causing significant soft-tissue defects in sun-exposed areas of skin and progressing to metastatic disease, which is usually associated with poor survival. The transforming growth factor-β (TGF-β) signalling pathway is known to play key regulatory roles in skin homeostasis and wound repair. Murine studies indicate that loss of TGF-β signalling is sufficient to drive cSCC, but conclusive evidence for a similar role in human cSCC remains elusive. Combining immunohistochemical and genetic studies of the TGF-β signalling pathway on human cSCC tissue, with a thorough examination of TGF-β responses in human primary cSCC cell lines in-vitro, this thesis aims to investigate the complex role of TGF-β signalling in human cSCC. An extensive tissue micro-array analysis demonstrated the consistent reduction of endogenous TGF-β signalling activity in human primary cSCC. This intriguingly correlated with higher risk thick tumours pathologically, indicating that TGF-β is likely to act primarily as a tumour suppressor in human cSCC and its reduction or loss may impart a significant growth advantage for cSCC tumour cells. This tumour suppressor effect was reflected in-vitro, whereby the majority of primary cSCC cell lines remained sensitive to TGF-β mediated growth arrest. Resistance to TGF-β tumour suppression was also identified, and mechanistically its main protagonist in cSCC cell lines appeared to be mutational loss of TGF-β receptors. Consolidating in-vitro findings, both whole exome sequencing and 454 pyrosequencing of human cSCC tissue revealed frequent functionally damaging mutations of both TGF-β type 1 and type 2 receptors, indicating that mutational loss of the TGF-β pathway may be a key driving event in human cSCC tumourigenesis. Perhaps most interestingly, mutational loss of TGF-β type 2 receptors in cSCC cell lines appeared to result in a novel pro-oncogenic dependence on TGF-β type 1 receptor kinase activity, highlighting not only the important paradoxical role of TGF-β mediated tumour promotion in cSCC, but also the potential for signalling crosstalk between alternative TGF-β superfamily members, namely Activin signalling, to drive tumourigenesis in the absence of active TGF-β signalling. Although further mechanistic studies are required to support this hypothesis, the mutational status of TGF-β type 2 receptors may not only provide a powerful prognostic tool for patients with cSCC, but also represent an important biomarker for the targeted use of TGF-β inhibitors in potentially aggressive disease where pro-tumourigenic responses could be driving disease progression.

No description available.

The transforming growth factor-ß (TGFß) pathway, including the bone morphogenetic protein (BMP), plays critical roles during embryogenesis and in adult tissue homeostasis. Hence, malfunctions in TGFß/BMP signalling result in several diseases. Signalling is initiated by ligand binding to cell surface receptor kinases, which phosphorylate and activate the R-SMAD transcription factors. R-SMADs translocate to the nucleus and regulate the transcription of hundreds of genes. The cellular responses to TGFß/BMP signals are tightly controlled and highly regulated. TGFß/BMP receptors and R-SMADs, as the intracellular mediators of TGFß/BMP ligands, are key targets for regulation to control duration and potency of signalling. Reversible ubiquitylation of R-SMADs and TGFß/BMP receptors is a key mechanism to control TGFß/BMP signalling. Several E3 ubiquitin ligases have been reported to regulate the turnover and activity of TGFß/BMP receptors and R-SMADs, however little is known about their cognate deubiquitylating enzymes (DUBs). A proteomic screen identified the DUBs OTUB1 and USP15 as potential novel regulators of the TGFß and BMP pathways respectively. Endogenous OTUB1 was recruited to the active phospho-SMAD2/3 complex only upon TGFß induction and OTUB1 had a crucial role in TGFß-mediated gene transcription and cellular migration. OTUB1 inhibited the ubiquitylation of phospho-SMAD2/3 by binding to and inhibiting the E2 ubiquitin-conjugating enzymes independently of its catalytic activity. Consequently, the depletion of OTUB1 in cells caused a rapid loss in levels of TGFß-induced phospho-SMAD2/3, which was rescued by the proteasomal inhibitor Bortezomib. These findings demonstrated a novel signal-induced phosphorylation-dependent recruitment of OTUB1 to its target. Hence, OTUB1 could be exploited as a target to intervene against diseases that are provoked by an imbalance in TGFß signalling. DUBs are highly regulated enzymes and recent reports have shed light into the molecular regulation OTUB1. The N-terminal region of OTUB1 harbours an ubiquitin binding domain, which is critical for its function to inhibit ubiquitylation. While investigating the role of OTUB1 in TGFß signalling, it became apparent that OTUB1 itself could be post-translationally modified by phosphorylation. Two phosphorylation sites at the OTUB1 N-terminal region have been identified by mass spectrometry. S18 of OTUB1 was phosphorylated in vitro by the type I TGFß receptor (ALK5), whereas S16 was phosphorylated by the constitutively active kinase CK2 in vitro and in vivo. Phosphorylation of the OTUB1 N-terminal region could affect its physiological function and requires further investigation. Although much is known about DUBs that target the type I TGFß receptor, no DUBs that target the type I BMP receptors had been identified. USP15 was identified in a proteomic screen as an interactor of SMAD6, which is a negative regulator of the BMP pathway. USP15 also binds to and deubiquitylates the type I BMP receptor (ALK3), thereby enhancing BMP signalling. Consequently, USP15 impacts BMP-induced SMAD1 phosphorylation, mouse osteoblastic differentiation and Xenopus embryogenesis. A proteomic approach identified O-GlcNAc transferase (OGT) as an interactor of SMAD2. SMADs have not been associated with O-GlcNAc modifications and the regulation of TGFß/BMP signalling by O-GlcNAcylation has not been investigated. Endogenous SMADs1-3 bound OGT and pulled down potential O-GlcNAc modified proteins. Furthermore, SMAD4 was possibly O-GlcNAcylated, which implies that O-GlcNAc modification could regulate TGFß/BMP signalling. Further investigation is needed to decipher the precise molecular mechanisms of this potential regulation.

The Par6-polarity pathway regulates breast cancer metastasis, and more recently has been shown to regulate transforming growth factor β (TGFβ)-induced apoptosis. Integrins may mediate the regulation of TGFβ-induced apoptosis by Par6, as they are key regulators of cell polarity, survival and death. First, we confirmed that blocking Par6 activation significantly inhibits TGFβ-induced apoptosis in both monolayer and three-dimensional NMuMG (Normal Murine Mammary Gland) cell culture models. TGFβ altered the expression of β1 and β4 integrins in NMuMG monolayers. In addition, TGFβ significantly reduced the basal localization of α6 and β4 integrins in NMuMG three-dimensional acini-like structures (p < 0.001), which was dependent on both Par6 and TGFβ receptor I (TβRI)/SMAD activation. We went on to show that the activities of integrin pro-survival signaling mediators, NF-κB and FAK, were altered in response to TGFβ, and that blocking Par6 activation in the Par6/S345A mutant maintained polarity and basal α6 and β4 integrin expression in the presence of TGFβ in NMuMG three-dimensional structures, in addition to a significant increase in FAK activation. This suggests that TGFβ alters the expression, localization and downstream signaling of integrins, which may contribute to TGFβ-induced apoptosis

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.

Das Mammakarzinom ist weltweit eine der häufigsten Malignomerkrankung der Frau. Im fortgeschrittenen Stadium der Erkrankung entwickeln bis zu 75 % der Patientinnen ossäre Metastasen. Basierend auf der Erkenntnis, dass GnRH-Analoga in vitro die Migration und Invasion von Mammakarzinomzellen während der Kokultur mit humanen Osteoblasten bzw. der Osteoblasten-ähnlichen Zellreihe MG-63 hemmen, sollten in dieser Arbeit die dem zugrundeliegenden molekularen Mechanismen näher betrachtet werden. Es ist bekannt, dass GnRH-Analoga in vielen verschiedenen GnRH-Rezeptor exprimierenden Tumorzelllinen die Wachstumsfaktor- und Wachstumsfaktorrezeptor-Expression beeinflussen und somit z.B. antiproliferative oder Apoptose-induzierende Effekte vermitteln. In dieser Arbeit wurde die humane Mammakarzinomzelllinie MCF-7 mit der osteoblasten-ähnlichen Zellreihe MG-63 kokultiviert. Zu bestimmten Zeitpunkten erfolgte die Behandlung der Mammakarzinomzellen mit drei verschiedenen GnRH-Analoga. Dem GnRH-I-Agonisten Triptorelin, dem GnRH-I-Antagonisten Cetrorelix und dem GnRH-II-Agonisten [D-Lysin]-GnRH-II. Neben einer nicht-kokultivierten Kontrolle jeder Zelllinie wurde eine nicht behandelte Kokulturkontrolle mitgeführt. Nach 48 h (bzw. nach 72 h od. 96 h) wurde der Versuch beendet und die mRNA-Expression von EGF, EGFR, TGF-beta und TGFBRI und -II in MCF-7 und MG-63 mittels PCR bestimmt. Zudem wurde die mRNA-Expression von EGF, EGFR und TGF-beta auch nach längerer Kokultur- und Behandlungszeit sowie unter Stressbedingungen beobachtet. Es hat sich gezeigt, dass es weder in MG-63 noch in MCF-7 signifikante Expressionsunterschiede von EGF, EGFR und TGF-beta im Vergleich zur Kokulturkontrolle gab. Dies hat sich auch weder im Zeitverlauf noch unter Stressbedingungen geändert. In MG-63 konnte man ebenfalls keine Expressionsunterschiede der TGFB-Rezeptoren beobachten. Dagegen konnte erstmals gezeigt werden, dass alle drei GnRH-Analoga die mRNA-Expression des TGFBR-I und -II während der Kokultur mit der osteoblasten-ähnlichen Zellreihe MG-63 signifikant hemmen. Während man in anderen Gewebetypen bereits eine verminderte Expression von TGFBR-I und -II nach Behandlung mit GnRH-Analoga nachweisen konnte, ist dies erstmals in einer Mammakarzinomzelllinie gelungen. Desweiteren kann man aus den vorliegenden Ergebnissen den Schluss ziehen, dass die verminderte Expression der TGFB-Rezeptoren eine Rolle in der bereits nachgewiesene GnRH-Analoga-vermittelten Migrations- und Invasionshemmung spielt.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute skeletal muscle injury leads to increases in activin B levels and when selectively neutralized with a monoclonal antibody, there is augmented skeletal muscle repair.

No description available.

Diabetic nephropathy represents the leading cause of end stage renal disease worldwide and requires a kidney transplant or dialysis to survive. The number of patients suffering from diabetes is expected to increase, thus the number of patients with diabetic nephropathy is expected to concomitantly increase. Current treatment for diabetic nephropathy is not sufficient to prevent disease progression in most patients thus there is a need to develop novel therapies to treat diabetic nephropathy. The earliest changes that occur during the pathogenesis of diabetes occur in the glomerulus. The mesangial cells are a subpopulation of cells in the glomerulus that are responsible for coordinating responses with other nearby cell types. Transforming growth factor (TGF)-β1 is a cytokine that mesangial cells secrete, and has been identified as a profibrotic factor during the pathogenesis of diabetic nephropathy. Concerns have been raised in the use of direct anti-TGF-β1 therapy due to adverse events (such as dyspepsia and diarrhea) and lack of efficacy of anti-TGF-β1 monoclonal antibody LY2382770 in patients with diabetic nephropathy. Thus, therapy aimed at modulating TGF-β1 expression or activity may be efficacious in the treatment of diabetic nephropathy while avoiding potential adverse effects. The hypothesis of this thesis is that SREBP-1 and cell surface GRP78 are novel regulators of TGF-β1 signaling in mesangial cells. Our first study aims to define a novel pathway by which SREBP-1 regulates TGF-β1 signaling in kidney mesangial cells. Our results indicate that SREBP-1 regulates the expression of the type I TGF-β1 receptor through its secretion in exosomes. Our second study expands on these findings and aims to determine if inhibition of SREBP in vivo with the inhibitor fatostatin may prevent diabetic nephropathy. Our results indicate that treatment with fatostatin does not prevent diabetic nephropathy, but accentuates kidney injury in non-diabetic mice. Preliminary results from our lab have indicated that under diabetic conditions, GRP78 is upregulated at the cell surface and may contribute to the activation of SREBP-1 in an ER-stress dependent mechanism. Our third study thus aims to characterize the expression of cell surface GRP78 in diabetic conditions, and to determine its pathological relevance in the development of diabetic nephropathy. Our results have established novel pathways by which TGF-β1 signaling is regulated in mesangial cells. This will assist in identification of novel therapeutic targets that may be of use in the treatment of diabetic nephropathy. / Thesis / Doctor of Philosophy (PhD) / Diabetic kidney disease is the leading cause of end stage renal disease and represents an important risk factor for mortality. The goal of this thesis is to understand and describe the pathways and mechanisms that contribute to the development of diabetic kidney disease in order to identify novel therapeutic targets. This thesis has identified the protein sterol regulatory element binding protein (SREBP)-1 and the cell surface presentation of another protein, the 78 kDa glucose regulated protein (GRP78), as contributors to diabetic kidney disease. Furthermore, this thesis has demonstrated that anti-SREBP therapy with the drug fatostatin did not prevent diabetic kidney disease. These studies show that while inhibiting SREBP-1 and cell surface GRP78 may be effective in the treatment of diabetic kidney disease, the drug fatostatin should not be used for treatment.