Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007. / Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and the ability to differentiate into multiple cell-lineages. The potential for the application of stem cells in clinical settings has had a profound effect on the future of regenerative medicine. However, to be of greater therapeutic use, selection of the most appropriate cell type, as well as optimisation of stem cell incorporation into the damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell population which mediate postnatal muscle growth. Following injury or in diseased conditions, these cells are activated and recruited for new muscle formation. In contrast, the potential of resident adult stem cell incorporation into the myocardium has been challenged and the response of cardiac tissue, especially to ischaemic injury, is scar formation. Following muscle damage, various growth factors and cytokines are released in the afflicted area which influences the recruitment and incorporation of stem cells into the injured tissue. Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation of cell growth and regeneration following activation and stimulation of receptor-signalling pathways. By improving the understanding of how TGF-β affects these processes, it is possible to gain insight into how the intercellular environment can be manipulated to improve stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis were to determine the effect of the three TGF-β isoforms on proliferation, differentiation, migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to possible improved mechanisms for muscle repair. The effect of short- and long-term treatment with all three TGF-β isoforms were investigated on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems. Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12 cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and together with total nuclear counts, used to assess the effect of TGF-β on myoblast proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms regulate differentiation, further analysis included determination of MyoD localisation and the rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of connexin-43 and MHC were analysed, together with the determination of embryoid body numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as well as fusion of C2C12 cells (Chapter 8). Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells, differentiation results, however, indicated that especially following long-term incubation, TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes. Similarly, myocyte migration and fusion were also negatively regulated following TGF-β treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with TGF-β1 could potentially enhance differentiation. Further research is however required in this area, especially since migration was greatly reduced in these cells. Taken together, results demonstrated variable effects following TGF-β treatment depending on the cell type and the duration of TGF-β application. Circulating and/or treatment concentrations of this growth factor could therefore be manipulated depending on the area of injury to improve regenerative processes. Alternatively, when selecting appropriate stem or progenitor cells for therapeutic application, the effect of the immediate environment and subsequent interaction between the two should be taken into consideration for optimal beneficial results.

O desmame precoce (DP) causa mudanças na mucosa gástrica, como o aumento da proliferação celular e da expressão do Fator de Crescimento Transformante (TGFa). Neste trabalho, avaliamos o papel desse peptídeo e seu receptor EGFR no controle do crescimento gástrico. Ratos com 15 dias de vida foram divididos em: amamentados (controle) e DP, no qual os filhotes foram separados da mãe e alimentados com pasta de ração. O DP aumentou o número de células marcadas para EGFR, acelerou a diferenciação de células mucosas do colo e elevou a expressão de mucina 6. A inibição do EGFR com AG1478 diminuiu a proliferação celular e o número de células mucosas do colo. Entre as proteínas envolvidas na sinalização de EGFR e ciclo celular, detectamos que o DP elevou os níveis de p-ERK1/2 e p-Src e não alterou p-Akt, p21 e p27. Nós sugerimos que o padrão alimentar influencia a proliferação e diferenciação no epitélio gástrico, e que TGFa/EGFR podem regular esses processos durante o desenvolvimento pós-natal, provavelmente por ativação das vias de sinalização de MAPK e Src. / Early weaning (EW) causes changes in the gastric mucosa, including the increase in cell proliferation and Transforming Growth Factor (TGFa). In the present study, we evaluated the role of this peptide and its receptor EGFR in the control of the gastric growth. 15-d-old rats were divided into two groups: suckling (control) and EW, in which the pups were separated from the dam and fed with powdered chow. EW increased the number of EGFR-positive cells, accelerated the differentiation of mucous neck cells and augmented the expression of mucin 6. EGFR inhibition with AG1478 decreased cell proliferation and the number of mucous neck cells. Among the proteins involved on EGFR signaling pathways and cell cycle, we found that EW increased the levels of p-ERK1/2 and p-Src, but did not change p-Akt, p21 and p27. We suggest that the diet pattern influences proliferation and differentiation in the gastric epithelium, and the TGFa/EGFR can regulate these processes throughout the postnatal development, probably by activating MAPK and Src signaling pathways.

The Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and member of the basic helix-loop-helix Per/ARNT/Sim (bHLH/PAS) family of chemosensors and developmental regulators. As a member of the PAS domain family of transcription factors responsive to exogenous signals, the AhR exerts influence on many processes relating to cellular fate. The activation of AhR is widely associated with toxic endpoints related to dioxin exposure. However, the AhR also activates endogenous gene programs related to development, cellular growth, and differentiation. The AhR is able to bind a variety of ligands, leading to a wide range of biological outcomes. Recent reports have shown that the AhR can mediate tumor suppressive effects. As a ligand-activated transcription factor, the AhR has the potential to actuate a variety of transcriptional programs that are dependent on the AhR ligand. Our central hypothesis is that AhR ligands can be identified that are capable of initiating tumor suppressive functions of the AhR. We utilized complementary cell-based and in silico virtual screening approaches to identify potential AhR ligands. We developed homology models of the AhR ligand-binding domain (LBD) for virtual ligand screening (VLS) of small molecule libraries. This led to the identification of new AhR ligands 5,7- dihydroxyflavanone!and 5-hydroxy-7-methoxyflavone. Additional small molecule libraries were screened in parallel that led to identification of flutamide as a putative AhR ligand. Flutamide is clinically approved for the treatment of prostate cancer due to its ability to antagonize androgen receptor mediated transcription. We investigated the biological effects of flutamide in AhR positive cancer cells that do not express the androgen receptor and found that flutamide inhibited the growth of HepG2 cells. Suppression of AhR expression reversed the anti-proliferative effects of flutamide. We tested 15 structural analogs of flutamide, including the flutamide metabolite 2-hydroxyflutamide for activation of AhR transcriptional activity. Flutamide is unique in its ability to activate the AhR, and suppresses hepatoma cell growth. These data suggests that flutamide-induced AhR transcriptional activity is required to initiate the tumor suppressive effects. We examined changes in cell cycle checkpoint proteins after flutamide treatment and discovered increased expression of cell cycle inhibitory proteins p27[superscript Kip1] and p15[superscript INK]. We also found that transforming Growth Factor β1 (TGFβ1), which regulates both p27[superscript Kip1] and p15[superscript INK], is upregulated by flutamide. We demonstrate that TGFβ1 is upregulated by flutamide in an AhR-dependent manner and is required for suppression of proliferation by flutamide. We identify specific and unique transcriptional signatures of the AhR upon activation by flutamide, that are distinct from the potent AhR agonist 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). In summary, we characterize flutamide as an AhR ligand and demonstrate its AhR-dependent tumor suppressive effects in hepatoma cells. We provide the first direct evidence that AhR regulates TGFβ signaling in a ligand dependent manner. We demonstrate that the AhR-induced downstream transcriptional signature and subsequent biological effects are specific to the AhR ligand. Our studies have broad impact for characterizing the AhR as a new therapeutic target in hepatocellular carcinoma. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from March 28, 2013 - March 28, 2015

The TGFβ family of cytokines are potent signalling molecules that regulate tissue development, inflammation and immunity. Previous studies in mice with a null mutation in the Tgfb1 gene (TGFβ1-/- mice) implicate a key role for TGFβ1 in male reproductive function. These mice show profound infertility due to an inability to copulate successfully, associated with reduced testosterone and sperm production. The focus of this project was to 1) further characterize mechanisms underpinning reproductive deficiency in male TGFβ1-/- mice, 2) identify a reliable physiological marker of TGFβ1 availability in vivo, and 3) to determine whether exogenous TGFβ1 administration influences TGFβ1 availability and restores fertility. To investigate the causes of unsuccessful copulation by TGFβ1-/- mice, penis morphometry was examined. Penile organ structure, as assessed by scanning electron microscopy, was comparable between genotypes however a superfluous epidermal covering that impeded penile spine protrusion was evident in TGFβ1-/- mice. The epidermal covering was not due to increased epithelial cell proliferation, as measured by Brdu labelling and immunohistology. Behavioural observations of erectile activity showed that TGFβ1-/- mice achieved spontaneous erections albeit at reduced frequency compared to TGFβ1+/+ mice. The efficacy of exogenous TGFβ1 replacement was evaluated by first identifying measures of in vivo TGFβ1 availability and/or function and selecting an effective route of administration. Serum TGFβ1 and testosterone levels were reliable discriminators of TGFβ1 genotype. Gene expression and phagocytic function of peritoneal macrophages revealed no differences between genotypes. Exogenous sources of TGFβ1 for replacement studies included colostrum, naturally occurring in breast milk and recombinant human latent TGFβ1 (rhLTGFβ1). Colostrum did not increase circulating levels and rhTGFβ1 injection caused only transient elevation of serum levels. Thus mini-osmotic pumps were used to deliver a constant supply of cytokine to TGFβ1-/- mice. The fertility status of TGFβ1-/- mice receiving exogenous TGFβ1 was investigated. Reproductive behaviour in response to normal receptive female mice was assessed twice during treatment, on day 7 and day 14. Blood, liver and reproductive tissues were collected at sacrifice. Circulating TGFβ1 was increased in TGFβ1 treated TGFβ1-/- mice above TGFβ1-/- control levels, although this did not affect circulating testosterone. Erectile activity and sperm production were unchanged. Videotaping behaviour with estrous females revealed that the TGFβ1+/+ mice successfully mounted and intromitted, unlike the TGFβ1-/- controls. The TGFβ1-/- mice receiving exogenous TGFβ1 displayed moderately enhanced mounting and intromission behaviour although this remained less frequent than in the TGFβ1+/+ controls. Ejaculation behaviour was not observed in any TGFβ1-/- mice regardless of TGFβ1 replacement, compared to TGFβ1+/+ controls where >90% mice displayed ejaculated. Modest improvement in the copulation activity of the TGFβ1-/- mice receiving exogenous TGFβ1 suggests that systemic TGFβ1 availability can influence reproductive performance in male TGFβ1-/- mice. However since fertility was not restored, locally produced TGFβ1 in the reproductive tract and/or hypothalamic pituitary axis are also implicated in regulating fertility. These findings advance our knowledge of the role of the TGFβ1 cytokine in male reproductive physiology and may have relevance for devising new treatments for infertility and erectile dysfunction in men. / Thesis (Ph.D.) - University of Adelaide, School of Paediatrics and Reproductive Health, 2008

O desmame precoce (DP) causa mudanças na mucosa gástrica, como o aumento da proliferação celular e da expressão do Fator de Crescimento Transformante (TGFa). Neste trabalho, avaliamos o papel desse peptídeo e seu receptor EGFR no controle do crescimento gástrico. Ratos com 15 dias de vida foram divididos em: amamentados (controle) e DP, no qual os filhotes foram separados da mãe e alimentados com pasta de ração. O DP aumentou o número de células marcadas para EGFR, acelerou a diferenciação de células mucosas do colo e elevou a expressão de mucina 6. A inibição do EGFR com AG1478 diminuiu a proliferação celular e o número de células mucosas do colo. Entre as proteínas envolvidas na sinalização de EGFR e ciclo celular, detectamos que o DP elevou os níveis de p-ERK1/2 e p-Src e não alterou p-Akt, p21 e p27. Nós sugerimos que o padrão alimentar influencia a proliferação e diferenciação no epitélio gástrico, e que TGFa/EGFR podem regular esses processos durante o desenvolvimento pós-natal, provavelmente por ativação das vias de sinalização de MAPK e Src. / Early weaning (EW) causes changes in the gastric mucosa, including the increase in cell proliferation and Transforming Growth Factor (TGFa). In the present study, we evaluated the role of this peptide and its receptor EGFR in the control of the gastric growth. 15-d-old rats were divided into two groups: suckling (control) and EW, in which the pups were separated from the dam and fed with powdered chow. EW increased the number of EGFR-positive cells, accelerated the differentiation of mucous neck cells and augmented the expression of mucin 6. EGFR inhibition with AG1478 decreased cell proliferation and the number of mucous neck cells. Among the proteins involved on EGFR signaling pathways and cell cycle, we found that EW increased the levels of p-ERK1/2 and p-Src, but did not change p-Akt, p21 and p27. We suggest that the diet pattern influences proliferation and differentiation in the gastric epithelium, and the TGFa/EGFR can regulate these processes throughout the postnatal development, probably by activating MAPK and Src signaling pathways.

Indiana University-Purdue University Indianapolis (IUPUI) / Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.