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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 2, 2005 as doi:10.1096/fj.05-4006fje. |
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,1
* Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada;
Department of Anatomy and Cell Biology, The George Washington University Medical Center, Washington, DC, USA; and
University of Toronto, Department of Medical Biophysics, Toronto, Ontario, Canada
1 Correspondence: University Health Network, 67 College St., Room 406, Toronto, Ontario, Canada M5G 2M1. E-mail: jmedin{at}uhnres.utoronto.ca
SPECIFIC AIMS
We used small synthetic molecules, generically termed chemical inducers of dimerization, to control specific interactions between modified receptor chimeras encoding the signaling domain of KDR. Using this system we sought to investigate the molecular pathways upon activation of this chimera in transduced human erythroleukemic TF1 cells by performing global gene expression analysis, functional and biochemical analyses of CID-treated and untreated cells.
PRINCIPAL FINDINGS
1. Activation of KDR induces growth in a factor-dependent human cell line
Human TF1 cells were transduced with a lentiviral vector encoding the tLNGFR cell surface tag, one copy of the FKBP dimerizing unit, and the cytoplasmic domain of KDR. From 144 isolated single cell clones, a total of 89 tLNGFR-positive cell clones were characterized. In short-term proliferation assays, 7 clones responded to AP20187 stimulation in a dose-dependent manner. Two clones (clones A5 and E5) responded unambiguously (Fig. 1
). Cell proliferation was observed with as low as 0.3 µM AP20187 and peaked at 3 µM. A5 cells were reliant on constant levels of AP20187 to maintain their viability. Nontransduced TF1 or enGFP-transduced TF1 cells were unresponsive to AP20187 stimulation.
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2. Activation of KDR induces the phosphorylation of Akt and Erk1/2
We immunoblotted cell lysates from AP20187-stimulated A5 cells with phospho-specific anti-Akt and Erk1/2 antibodies. Although low constitutive Akt-kinase phosphorylation was observed in unstimulated A5 cells, Akt was maximally phosphorylated 10–20 min after stimulation. More strikingly, AP20187-mediated stimulation of KDR showed robust Erk1/2 activation (max. 10–30 min poststimulation), with legible Erk1/2 background activity in unstimulated cells.
3. KDR-induced gene expression analysis
Gene expression profiling was performed on stimulated A5 cells using Affymetrix high-density oligonucleotide arrays. To focus on mRNAs that were significantly modulated, we performed 3 x 3 comparisons (3 treated vs. 3 controls), with expression cut-off values of twofold or greater. Gene profiling expression analysis indicated a significant increase in mRNAs associated with cell processes including acute heat shock. Altered genes included Hsp105, SLC20A1, Gadd45a, and two polymorphic transcripts encoding for Hsp70.
4. KDR prevents apoptosis and premature cell senescence via Hsp70 and Hsp105
We stimulated A5 cells with AP20187 and KNK437, a specific inhibitor of Hsp70 and Hsp105. In apoptosis assays, KNK437 induced a 66% increase in the proportion of Annexin V-positive cells vs. untreated cells. In contrast, a significant reduction in the proportion of apoptotic cells, comparable to AP20187-treated controls, was seen with cells incubated with KNK437 plus AP20187 (Fig. 2
A). We investigated the effects of Hsp70 and Hsp105 on cell proliferation. We observed that AP20187 exerted a proliferative effect that was 
9-fold higher in comparison to untreated and KNK437-treated cells. In AP20187 plus KNK437-treated cells, the proliferative response was markedly diminished in comparison to AP20187-treated cells, however the proliferative response in these cultures was significantly higher (4-fold) in comparison to KNK437-treated cells (Fig. 2B
).
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5. Akt is an upstream target of Hsp70 and Hsp105
Having shown that KDR transmits signals via PI-3K/Akt and MAP kinase pathways, we hypothesized that Hsp70 or Hsp105 chaperone activity may also regulate members belonging to these signaling families. We analyzed lysates of A5 cells that were treated with AP20187 and KNK437 and immunoblotted using antibodies to Akt and Erk1/2. Akt was present in abundant levels in AP20187-stimulated cells. When cells were coincubated with AP20187 and increasing concentrations of KNK437, a dose-dependent decrease in Akt production was observed. Collectively, Fig. 3
outlines a diagrammatic representation of our findings.
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CONCLUSIONS
Cell expansion using CID to regulate receptor dimerization and activation can augment the treatment of genetic and acquired disorders. CID-induced aggregation of receptors is also a promising tool to further understand receptor interactions and transcriptional changes in associated genes that follow from these events in a specific and selective manner. While some publications have reported the efficiency of using such systems to regulate cytokine receptor function in cultured cells and in whole animals, more analyses are required to garner a greater understanding of the intracellular circuitry that control these processes. We designed our experiments to begin to address this by establishing cloned cell lines overexpressing an engineered KDR growth switch that responded to AP20187 and then by characterizing dimerization effects using biochemical and global gene microarray analyses.
We chose to overexpress a chimeric KDR construct in human TF1 erythroleukemia cells. We hypothesize that this cell line is as an appropriate target to initially test these effects as it shares some features with primary hematopoietic cells, including the ability to self-renew, differentiate into multiple lineages, and express early hematopoietic markers such as CD34.
It has been shown that VEGF stimulation of KDR induces endothelial cell proliferation. VEGF activation of KDR in acute myeloid leukemia cells supports their survival and proliferation. In agreement with these studies, we demonstrated that TF1 cell clones expressing a KDR growth factor switch specifically proliferated in response to AP20187 in a dose-dependent manner.
In addition to its proliferative effects, stimulation of KDR has also been shown to induce endothelial and hematopoietic cell survival. Our study showed that activation of a KDR growth switch contributed to anti-apoptotic effects in A5 cells. A potential mechanism by which this is achieved could involve the repression of FOXO3a phosphorylation via Akt. Recently a report published by Rosas and colleagues documented that IL-5 deprivation from TF1 cells resulted in the dephosphorylation of this transcription factor that lead to an increase in Bim protein production that initiated programmed cell death.
Complex networks of signaling pathways, in particular MAPK and PI-3K cascades, have been shown to mediate proliferative and survival responses via activated KDR. Implementation of chimeric VEGFR constructs that are activated by small-molecule dimerizers has shown activation of these cell signaling mediators. Congruent with this, we demonstrated that AP20187-activation of chimeric KDR results in the phosphorylation of Erk1/2 and Akt substrates in A5 cells.
Microarray analysis of stimulated TF1-L1FK A5 cells identified up-regulated genes associated with cell survival, proliferation, cytoskeletal remodeling, and migration. Among genes uniquely expressed, Hsp70 and Hsp105 might be underlying mediators that are regulating the functional effects via activated KDR. Hsp70 has a broad spectrum of functions that regulate a variety of cell processes including cell cycle control, proliferation, and differentiation. Hsp70 and co-chaperones interact with signaling molecules, including nuclear hormone receptors, intracellular kinases, and regulators of cell cycle and death. In the present study, AP20187-stimulated A5 cells manifested an increased resistance to KNK437-induced premature cell senescence. Hsp70 transiently associates with important regulators of the cell cycle including cyclin B1, c-myc, pRb, p53, and p27/Kip1. Signaling intermediates involved in cell proliferation such as Mos interact with Hsp70. Along with prominent signal transduction pathways associated with MAPK cascades, we hypothesize that the stabilization of these molecules via Hsp70 could be acting in a synergistic manner promoting cell growth. We demonstrated that activation of the KDR growth switch in A5 cells antagonizes the apoptotic-inducing effects of KNK437. Mechanisms here may include the binding of Hsp70 to APAF-1 and/or Bcl-2, which are involved in the regulation of cell survival. These potential mechanisms suggest that KDR can play a role in leukemic conversion of hematopoietic cells. Hence these results may explain how mitogenic effects imparted by VEGF on responsive leukemic subsets may be coordinated by Hsp70. More important, these outcomes suggest that Hsp70 may be a possible target for therapy.
Hsp70 transcription is controlled by heat shock transcription factor 1, which is activated by Ras-mediated initiation of the PI-3K cascade. In the current study, blockade of Hsp70 lead to the marked reduction in Akt protein production. A similar decrease of Akt production has been demonstrated using Hsp90 inhibitors in Flt3-expressing leukemias. In contrast, the quantity of Erk1/2 in our study was unaffected. This finding suggests that post-translational regulation of Akt involving Hsp70 may contribute to PI-3K signaling events and could provide a key mechanism of positively regulating Akt activity.
It has been reported that Hsp70 promotes differentiation and prevents apoptosis in HL60 cells. Although no evidence for differentiation was demonstrated in our stimulated A5 cells, many differentiation-associated transcripts were modulated including Id2, EPS8, MAPK6, and Pim1. Demonstrating a proliferative growth advantage in transduced primary cells maybe more difficult given the potential this receptor has for inducing cell differentiation. Identification and removal of domains responsible for differentiation may improve signaling events that bias for hematopoietic cell division.
Our observations describe an experimental approach to decipher signaling and molecular events associated with an inducible KDR growth switch. Results complement and significantly extend previously published data and understanding of KDR signaling. The application of functional, biochemical and microarray analyses in our study has identified that a plethora of genes are orchestrated in response to specific KDR activation by pharmacological manipulation, which leads to cell proliferation and maintenance of viability. Further study may refine definition of signals required to augment transduced cell numbers, which can improve gene therapy outcomes.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4006fje; doi: 10.1096/fj.05-4006fje
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