Related Experiment Videos
Molecular mechanism of transforming growth factor beta-mediated cell-cycle modulation in primary human CD34(+)
Mo A Dao1, Joseph Hwa, Jan A Nolta
1Division of Research Immunology/Bone Marrow Transplantation, Children's Hospital Los Angeles, and Department of Pediatrics, University of Southern California School of Medicine, Los Angeles, CA 90027, USA.
Abstract:
The mechanisms by which transforming growth factor beta (TGF-beta) exerts a negative effect on cell-cycle entry in primary human hematopoietic stem/progenitor cells were examined at the molecular and cellular levels. After treatment of primary human CD34+ progenitors with TGF-beta there was a decrease in the levels of cyclin D2 protein and an increase in levels of the cyclin-dependent kinase inhibitor (CDKI) p15 as compared to the levels in untreated cells. The converse was true after addition of neutralizing anti-TGF-beta antibody. Administration of TGF-beta to CD34+ cells in the presence of cytokines prevented retinoblastoma protein (pRb) phosphorylation, which occurred in the same cells treated with cytokines alone or cytokines and anti-TGF-beta antibody. Neutralization of TGF-beta during 24 to 48 hours of culture with cytokines significantly increased the number of colony-forming progenitors, but did not modulate the human stem cell pool, as measured in 6- to 12-month xenotransplantation assays. Equivalent numbers of human B, T, and myeloid cells were obtained after transplantation of cells treated with or without neutralization of TGF-beta.
Insights
Transforming growth factor beta (TGF-beta) inhibits cell-cycle entry in hematopoietic stem cells by altering cyclin D2 and p15 levels. Neutralizing TGF-beta boosts progenitor cell formation without affecting the overall stem cell pool.
Area of Science:
- Hematology
- Cell Biology
- Molecular Biology
Background:
- Transforming growth factor beta (TGF-beta) is known to regulate cell proliferation and differentiation.
- Its precise mechanisms in primary human hematopoietic stem/progenitor cells require further elucidation.
Purpose of the Study:
- To investigate the molecular mechanisms underlying TGF-beta's negative regulation of cell-cycle entry in human hematopoietic stem/progenitor cells.
- To assess the impact of TGF-beta modulation on progenitor cell expansion and stem cell pool maintenance.
Main Methods:
- Primary human CD34+ hematopoietic progenitors were treated with TGF-beta or neutralizing anti-TGF-beta antibody.
- Levels of cyclin D2 protein and cyclin-dependent kinase inhibitor (CDKI) p15 were analyzed.
- Retinoblastoma protein (pRb) phosphorylation was assessed in cells cultured with cytokines.
- Xenotransplantation assays were performed to evaluate the stem cell pool and cell differentiation.
Main Results:
- TGF-beta treatment decreased cyclin D2 protein and increased p15 levels in CD34+ progenitors.
- TGF-beta prevented pRb phosphorylation in the presence of cytokines.
- Neutralization of TGF-beta increased colony-forming progenitors but did not alter the stem cell pool.
- Transplantation assays showed equivalent B, T, and myeloid cell production regardless of TGF-beta neutralization.
Conclusions:
- TGF-beta inhibits cell-cycle entry in human hematopoietic stem/progenitor cells via modulation of cyclin D2 and p15.
- TGF-beta's action on pRb phosphorylation is a key mechanism in cell-cycle regulation.
- Targeting TGF-beta can enhance progenitor cell expansion without compromising the long-term stem cell pool.