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Smad signaling in hematopoietic stem cell biology

Karlsson, Göran LU (2007)
Abstract
Hematopoietic stem cells (HSCs) are primitive tissue-specific somatic stem cells, responsible for the maintenance and replenishment of the bone marrow (BM) and subsequently the entire blood system. The never-ending ability to differentiate into all the mature hematopoietic lineages makes these HSCs attractive candidates for use in future regenerative medicine. HSCs are balanced between different fate options through a fine-tuned orchestra of regulatory factors. The large superfamily of multifunctional growth factors known as the transforming growth factor 's (TGF-?s) has been suggested to play a role in this regulation. Despite acting through a seemingly simple signaling circuit, known as the Smad pathway, members of the TGF-? superfamily... (More)
Hematopoietic stem cells (HSCs) are primitive tissue-specific somatic stem cells, responsible for the maintenance and replenishment of the bone marrow (BM) and subsequently the entire blood system. The never-ending ability to differentiate into all the mature hematopoietic lineages makes these HSCs attractive candidates for use in future regenerative medicine. HSCs are balanced between different fate options through a fine-tuned orchestra of regulatory factors. The large superfamily of multifunctional growth factors known as the transforming growth factor 's (TGF-?s) has been suggested to play a role in this regulation. Despite acting through a seemingly simple signaling circuit, known as the Smad pathway, members of the TGF-? superfamily have various cellular effects depending on context. Several attempts have been made to link Smad signaling and HSC biology but the complexity of this system and its critical role for embryonic development have limited these studies to cell cultures. In this thesis, we have completely disrupted Smad signaling in adult HSCs in vivo by overexpressing the inhibitory Smad7 and by eliminating the common Smad4, using a conditional knockout model. The former approach prevents activation of R-Smads but leaves Smad4 intact whereas the latter model eliminates the physiological function of Smad4, the key downstream molecule where all Smad pathways converge. Additionally, we sought to identify gene targets for TGF-? by means of gene expression profiling analysis on activin receptor-like kinase (Alk) 5-/- fibroblast cell lines.



Smad7 overexpressing HSCs could long-term reconstitute the hematopoietic system of lethally irradiated recipients and displayed normal lineage distribution. However, following secondary transplantation, Smad7 overexpressing cells had a significantly increased regenerative capacity as compared to control transduced cells. In contrast, competitive repopulation capacity of Smad4-/- HSCs was significantly impaired and Smad4 null cells were completely out-competed in secondary hosts. Intriguingly, enforced expression of Smad7 in Smad4 null HSCs failed to expand HSCs in vivo, suggesting that the Smad7 HSC expansion phenotype is dependent on physiological concentrations of Smad4 and implicating that Smad4 is involved in relaying self-renewal signals from additional regulatory circuits. Moreover, gene expression profiling of murine embryonic fibroblasts, deficient in the TGF-? receptor revealed that TGF-? signaling occurs exclusively through Alk5 in these cells. Furthermore, we obtained a database of 369 differentially expressed genes which will be used to develop techniques for further studies of Smad signaling and cross-talk in HSCs. Taken together, our results have not only confirmed conventional theories regarding the role of TGF-? in HSC biology but also opened new exciting questions concerning the complexity and context dependency of TGF-? superfamily signaling, challenging our understanding of the Smad circuit in general. (Less)
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author
supervisor
opponent
  • Professor Dzierzak, Elaine, Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlan
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Smad7, Smad4, microarray, retroviral-mediated gene expression, conditional knockout, transforming growth factor beta, smad signaling, hematopoiesis, hematopoietic stem cells, Alk5, Medicine (human and vertebrates), Medicin (människa och djur)
pages
114 pages
publisher
Molecular Medicine and Gene Therapy, Lund University
defense location
Segerfalksalen, Wallenberg Neurocenter, Sölvegatan 17
defense date
2007-09-22 09:00
ISSN
1652-8220
ISBN
978-91-85897-02-5
language
English
LU publication?
yes
id
6be90f94-706b-4bc2-9af8-3d995c9aa06f (old id 549004)
date added to LUP
2007-11-13 08:12:39
date last changed
2016-09-19 08:44:57
@phdthesis{6be90f94-706b-4bc2-9af8-3d995c9aa06f,
  abstract     = {Hematopoietic stem cells (HSCs) are primitive tissue-specific somatic stem cells, responsible for the maintenance and replenishment of the bone marrow (BM) and subsequently the entire blood system. The never-ending ability to differentiate into all the mature hematopoietic lineages makes these HSCs attractive candidates for use in future regenerative medicine. HSCs are balanced between different fate options through a fine-tuned orchestra of regulatory factors. The large superfamily of multifunctional growth factors known as the transforming growth factor 's (TGF-?s) has been suggested to play a role in this regulation. Despite acting through a seemingly simple signaling circuit, known as the Smad pathway, members of the TGF-? superfamily have various cellular effects depending on context. Several attempts have been made to link Smad signaling and HSC biology but the complexity of this system and its critical role for embryonic development have limited these studies to cell cultures. In this thesis, we have completely disrupted Smad signaling in adult HSCs in vivo by overexpressing the inhibitory Smad7 and by eliminating the common Smad4, using a conditional knockout model. The former approach prevents activation of R-Smads but leaves Smad4 intact whereas the latter model eliminates the physiological function of Smad4, the key downstream molecule where all Smad pathways converge. Additionally, we sought to identify gene targets for TGF-? by means of gene expression profiling analysis on activin receptor-like kinase (Alk) 5-/- fibroblast cell lines.<br/><br>
<br/><br>
Smad7 overexpressing HSCs could long-term reconstitute the hematopoietic system of lethally irradiated recipients and displayed normal lineage distribution. However, following secondary transplantation, Smad7 overexpressing cells had a significantly increased regenerative capacity as compared to control transduced cells. In contrast, competitive repopulation capacity of Smad4-/- HSCs was significantly impaired and Smad4 null cells were completely out-competed in secondary hosts. Intriguingly, enforced expression of Smad7 in Smad4 null HSCs failed to expand HSCs in vivo, suggesting that the Smad7 HSC expansion phenotype is dependent on physiological concentrations of Smad4 and implicating that Smad4 is involved in relaying self-renewal signals from additional regulatory circuits. Moreover, gene expression profiling of murine embryonic fibroblasts, deficient in the TGF-? receptor revealed that TGF-? signaling occurs exclusively through Alk5 in these cells. Furthermore, we obtained a database of 369 differentially expressed genes which will be used to develop techniques for further studies of Smad signaling and cross-talk in HSCs. Taken together, our results have not only confirmed conventional theories regarding the role of TGF-? in HSC biology but also opened new exciting questions concerning the complexity and context dependency of TGF-? superfamily signaling, challenging our understanding of the Smad circuit in general.},
  author       = {Karlsson, Göran},
  isbn         = {978-91-85897-02-5},
  issn         = {1652-8220},
  keyword      = {Smad7,Smad4,microarray,retroviral-mediated gene expression,conditional knockout,transforming growth factor beta,smad signaling,hematopoiesis,hematopoietic stem cells,Alk5,Medicine (human and vertebrates),Medicin (människa och djur)},
  language     = {eng},
  pages        = {114},
  publisher    = {Molecular Medicine and Gene Therapy, Lund University},
  school       = {Lund University},
  title        = {Smad signaling in hematopoietic stem cell biology},
  year         = {2007},
}