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Cited by 29Cytokine Signaling Pathways and InteractionsProtein Kinase Regulation and GTPase SignalingProtein Tyrosine Phosphatases

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The haematopoietic GTPase RhoH modulates IL3 signalling through regulation of STAT activity and IL3 receptor expression

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Gündogdu, Mehtap S, Liu, He, Metzdorf, Daniela, Hildebrand, Dagmar, Aigner, Michael, Aktories, Klaus, Heeg, Klaus, Kubatzky, Katharina F·2010·Molecular Cancer
DOI 10.1186/1476-4598-9-225PMC PMC2936343
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Abstract

Background: RhoH is a constitutively active member of the family of Rho GTPases. Its expression is restricted to the haematopoietic lineage, where it serves as a positive regulator for T cell selection and mast cell function and as a negative regulator for growth-related functions in other lineages. Here, we examined the activation of signal transducer and activator of transcription (STAT) proteins in response to stimulation with interleukin 3 (IL3). Results: Using the murine IL3-dependent cell line BaF3 we investigated the influence of RhoH protein expression levels on IL3-mediated cellular responses. RhoH overexpressing cells showed lower sensitivity to IL3 and decreased STAT5 activation. SiRNA-mediated repression of RhoH gene expression led to an increase in proliferation and STAT5 activity which correlated with an increased number of IL3 receptor α chain molecules, also known as CD123, expressed at the cell surface. Interestingly, these findings could be reproduced using human THP-1 cells as a model system for acute myeloid leukaemia, where low RhoH levels are known to be an unfavourable prognostic marker. Overexpression of RhoH on the other hand caused an induction of STAT1 activity and western blot analysis revealed that activated STAT1 is phosphorylated on Tyr701. STAT1 is known to induce apoptosis or cell cycle arrest and we detected an upregulation of cyclin-dependent kinase inhibitors (CDKI) p21 Cip1 and p27 Kip1 in RhoH overexpressing BaF3 cells. Conclusions: We propose that RhoH functions as a negative regulator for IL3-induced signals through modulation of the JAK-STAT pathway. High levels of RhoH allow the IL3-dependent activation of STAT1 causing decreased proliferation through upregulation of p21 Cip1 and p27 Kip1 . Low RhoH levels on the other hand led to an upregulation of IL3-dependent cell growth, STAT5 activity and an increase of CD123 surface expression, linking RhoH to a CD123/STAT5 phenotype that has been described in AML patients.

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TL;DR

RhoH expression level inversely regulates IL3-driven proliferation in BaF3 cells by modulating STAT1/STAT5 activity and CD123/IRF-1 expression. Low RhoH promotes a STAT5/CD123 phenotype also seen in THP-1 AML cells, while high RhoH activates STAT1 and cell-cycle inhibitors.

Key findings

  • In BaF3 cells, RhoH overexpression reduced IL3-dependent proliferation to app. 30% of control, whereas siRhoH increased proliferation to 160%; the effect was specific to IL3, as EpoR and EpoR-RhoH cells showed no growth difference.
  • RhoH overexpression decreased IL3-induced STAT5 phosphorylation to 50% and increased STAT1 Tyr701 phosphorylation app. two-fold; siRhoH increased STAT5 phosphorylation 1.3-fold, with no change in total STAT5 protein.
  • RhoH overexpression increased p21Cip1 and p27Kip1 mRNA 55- and 40-fold, respectively, and protein levels 2-fold and 1.3-fold; no significant changes were detected in siRhoH cells.
  • siRhoH BaF3 cells expressed app. 25% more surface CD123 and app. 200-fold higher IRF-1 mRNA, while RhoH overexpression decreased CD123 by app. 50%; in THP-1 cells, RhoH overexpression decreased CD123 by 50% and IRF-1 expression 15-fold.

Methods

  • Murine IL3-dependent BaF3 proB cells were retrovirally transduced to overexpress RhoH or to express siRhoH, with empty-vector controls; human THP-1 acute monocytic leukaemia cells were transiently transfected with HA-RhoH or vector.
  • Proliferation/viability was measured by Cell Titer Glo ATP assay, apoptosis by Annexin V/PI FACS, STAT phosphorylation by intracellular FACS and immunoprecipitation/western blot, CD123 surface expression by FACS, and mRNA by quantitative real-time PCR with GAPDH as reference.
  • Statistical significance was assessed by paired t-test; data are presented as mean ± SD, with most experiments performed at least in three independent experiments, though some quantifications used n = 2.

Limitations

  • Stated: the exact mechanism by which RhoH negatively regulates Rac1 remains to be elucidated; RhoH-deficient animals showed no obvious haematopoietic defect and did not develop lymphomas, suggesting compensation by other cytokines and a limit of the animal model.
  • Stated/evident: some supporting data are not shown (IL3-treated bone marrow cells from wildtype vs RhoH-deficient mice; siRhoH survival after cytokine deprivation/readdition), and several experiments have low n (n = 2 for some qPCR/western quantifications).
  • Evident: the proposed STAT5→IRF-1→CD123 axis is correlative; no direct manipulation of IRF-1 or STAT5 is reported, and THP-1 findings rely on transient transfection.

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Crossref · 10.1186/1476-4598-9-225

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Figures

Figure 1 RhoH regulates IL3-induced proliferation. (A) RhoH mRNA expression levels in control cells, RhoH or siRhoH-transduced BaF3 cells were detected by quantitative real-time PCR using RhoH specific primers and GAPDH as a reference gene. Statistical significance was analysed using the 's t-test (mean ± SD, n = 2 and *P ≤ 0.05; **P ≤ 0.01). (B) FACS analysis of control cells transduced with the empty vector (pMX-IRES-CD4-Puro) or RhoH (pMX-IRES-CD4-Puro RhoH) -transduced BaF3 cells for human CD4 expression after puromycin selection. Parental BaF3 cells were used as a negative control. (C) Dose response curve of control cells, RhoH and siRhoH BaF3 cells cultivated with increasing concentrations of IL3. Viability was determined by quantification of cellular ATP in a Cell Titer Glo assay. Results shown are from one representative experiment performed in triplicates ± SD. (D) Dose response curve of BaF3 cells, BaF3-EpoR cells and BaF3-EpoR cells overexpressing RhoH cultivated with increasing concentrations of Epo. Viability was determined by quantification of cellular ATP in a Cell Titer Glo assay. Results shown are from one representative experiment performed in triplicates ± SD.
Figure 1 RhoH regulates IL3-induced proliferation. (A) RhoH mRNA expression levels in control cells, RhoH or siRhoH-transduced BaF3 cells were detected by quantitative real-time PCR using RhoH specific primers and GAPDH as a reference gene. Statistical significance was analysed using the 's t-test (mean ± SD, n = 2 and *P ≤ 0.05; **P ≤ 0.01). (B) FACS analysis of control cells transduced with the empty vector (pMX-IRES-CD4-Puro) or RhoH (pMX-IRES-CD4-Puro RhoH) -transduced BaF3 cells for human CD4 expression after puromycin selection. Parental BaF3 cells were used as a negative control. (C) Dose response curve of control cells, RhoH and siRhoH BaF3 cells cultivated with increasing concentrations of IL3. Viability was determined by quantification of cellular ATP in a Cell Titer Glo assay. Results shown are from one representative experiment performed in triplicates ± SD. (D) Dose response curve of BaF3 cells, BaF3-EpoR cells and BaF3-EpoR cells overexpressing RhoH cultivated with increasing concentrations of Epo. Viability was determined by quantification of cellular ATP in a Cell Titer Glo assay. Results shown are from one representative experiment performed in triplicates ± SD.CC BY · PubMed Central
Figure 2 RhoH expression triggers IL3-dependent STAT1 activation. (A) Control cells, siRhoH and RhoH-transduced BaF3 cells were depleted of cytokine and FBS for 3 h prior to stimulation with IL3. Phosphorylated STAT1 levels were measured with intracellular FACS staining using a FITC-labelled pTyr-STAT1 specific antibody. One representative result of three independent experiments is shown. (B) Control and RhoH cells were starved for 3 hours prior to stimulation with 50 ng/ml IL3. Cells were lysed, STAT1 was immunoprecipitated and tyrosine phosphorylated STAT1 and total STAT1 were detected by enhanced chemiluminescence. Quantification of pSTAT1 levels was done after normalisation to STAT1 levels in cells transduced with the empty vector and is presented as induction of phosphorylation compared to the corresponding unstimulated sample. Statistical significance was analysed using the 's t-test (mean ± SD, n = 3; ***P ≤ 0.005). (D) Apoptosis was induced in control cells, siRhoH or RhoH-transduced cells by cultivation in the absence of IL3 (overnight), treatment with 1 μM staurosporine (3 h) or with 1 μM doxorubicine (12 h) prior to Annexin V/PI staining and FACS analysis (mean ± SD, n = 3).
Figure 2 RhoH expression triggers IL3-dependent STAT1 activation. (A) Control cells, siRhoH and RhoH-transduced BaF3 cells were depleted of cytokine and FBS for 3 h prior to stimulation with IL3. Phosphorylated STAT1 levels were measured with intracellular FACS staining using a FITC-labelled pTyr-STAT1 specific antibody. One representative result of three independent experiments is shown. (B) Control and RhoH cells were starved for 3 hours prior to stimulation with 50 ng/ml IL3. Cells were lysed, STAT1 was immunoprecipitated and tyrosine phosphorylated STAT1 and total STAT1 were detected by enhanced chemiluminescence. Quantification of pSTAT1 levels was done after normalisation to STAT1 levels in cells transduced with the empty vector and is presented as induction of phosphorylation compared to the corresponding unstimulated sample. Statistical significance was analysed using the 's t-test (mean ± SD, n = 3; ***P ≤ 0.005). (D) Apoptosis was induced in control cells, siRhoH or RhoH-transduced cells by cultivation in the absence of IL3 (overnight), treatment with 1 μM staurosporine (3 h) or with 1 μM doxorubicine (12 h) prior to Annexin V/PI staining and FACS analysis (mean ± SD, n = 3).CC BY · PubMed Central
Figure 3 RhoH upregulates p21Cip1 and p27Kip1 expression. (A) Quantification of p21Cip1 and p27Kip1 mRNA expression levels by quantitative SYBR-green PCR in control cells, RhoH and siRhoH-transduced cells. Data were normalised to mRNA levels detected in control cells compared to GAPDH. Statistical significance was analysed using the 's t-test (mean ± SD, n = 3; *P ≤ 0.05). (B, C) Whole cell lysates of control cells, siRhoH and RhoH expressing cells were separated by 4-20% gradient SDS-PAGE and protein expression was detected by antibodies against p21Cip1 and p27Kip1. Protein levels were normalised to β-actin expression (lower panel). Quantification and statistical significance were performed using the 's t-test (mean ± SD, n = 2; *P ≤ 0.05).
Figure 3 RhoH upregulates p21Cip1 and p27Kip1 expression. (A) Quantification of p21Cip1 and p27Kip1 mRNA expression levels by quantitative SYBR-green PCR in control cells, RhoH and siRhoH-transduced cells. Data were normalised to mRNA levels detected in control cells compared to GAPDH. Statistical significance was analysed using the 's t-test (mean ± SD, n = 3; *P ≤ 0.05). (B, C) Whole cell lysates of control cells, siRhoH and RhoH expressing cells were separated by 4-20% gradient SDS-PAGE and protein expression was detected by antibodies against p21Cip1 and p27Kip1. Protein levels were normalised to β-actin expression (lower panel). Quantification and statistical significance were performed using the 's t-test (mean ± SD, n = 2; *P ≤ 0.05).CC BY · PubMed Central
Figure 4 Low RhoH expression levels enhance STAT5 activity. (A) Control cells, siRhoH and RhoH expressing cells were starved for 3 h in the absence of cytokine and FBS before stimulation with 50 ng/ml IL3. Cells were lysed and STAT5 was immunoprecipitated. Tyrosine phosphorylated STAT5 and total STAT5 were detected by enhanced chemiluminescence using specific antibodies. Quantification of pSTAT5 levels was done after normalisation to total STAT5 levels in control cells and is presented as induction of phosphorylation compared to the corresponding unstimulated sample. Statistical significance was analysed using the 's t-test (mean ± SD, n = 2; *P ≤ 0.05). (B) To verify equal expression levels of STAT5 protein in all cell lines, STAT5 was detected from whole cell lysates and quantified after normalisation to β-actin levels of control cells (mean ± SD, n = 3). (C) pSTAT5 levels were measured by intracellular FACS staining at 0 and 10 min of stimulation of previously starved cells with 50 ng/ml IL3. Representative graphs from one out of three independently performed experiments are shown. (D) CD123 surface expression of control cells, siRhoH and RhoH cells determined by FACS analysis using a CD123-PE antibody. The left panel shows a typical example and the right panel summarises the data of the mean fluorescence signal and the statistical analysis (mean ± SD, n = 3, *P ≤ 0.05). (E) Quantification of IRF-1 mRNA expression levels of control cells and siRhoH-transduced cells was done by quantitative real-time PCR using GAPDH as a reference gene. Data were normalised to the expression in control cells and are presented as fold induction of the IRF-1 gene compared to control cells (mean ± SD, n = 3; *P ≤ 0.05).
Figure 4 Low RhoH expression levels enhance STAT5 activity. (A) Control cells, siRhoH and RhoH expressing cells were starved for 3 h in the absence of cytokine and FBS before stimulation with 50 ng/ml IL3. Cells were lysed and STAT5 was immunoprecipitated. Tyrosine phosphorylated STAT5 and total STAT5 were detected by enhanced chemiluminescence using specific antibodies. Quantification of pSTAT5 levels was done after normalisation to total STAT5 levels in control cells and is presented as induction of phosphorylation compared to the corresponding unstimulated sample. Statistical significance was analysed using the 's t-test (mean ± SD, n = 2; *P ≤ 0.05). (B) To verify equal expression levels of STAT5 protein in all cell lines, STAT5 was detected from whole cell lysates and quantified after normalisation to β-actin levels of control cells (mean ± SD, n = 3). (C) pSTAT5 levels were measured by intracellular FACS staining at 0 and 10 min of stimulation of previously starved cells with 50 ng/ml IL3. Representative graphs from one out of three independently performed experiments are shown. (D) CD123 surface expression of control cells, siRhoH and RhoH cells determined by FACS analysis using a CD123-PE antibody. The left panel shows a typical example and the right panel summarises the data of the mean fluorescence signal and the statistical analysis (mean ± SD, n = 3, *P ≤ 0.05). (E) Quantification of IRF-1 mRNA expression levels of control cells and siRhoH-transduced cells was done by quantitative real-time PCR using GAPDH as a reference gene. Data were normalised to the expression in control cells and are presented as fold induction of the IRF-1 gene compared to control cells (mean ± SD, n = 3; *P ≤ 0.05).CC BY · PubMed Central
Figure 5 The AML cell line THP-1 displays an siRhoH phenotype. (A) THP-1 cells were either transfected with HA-RhoH or the empty vector, lysed and separated by SDS-PAGE. Detection of HA-RhoH was performed with an HA-specific antibody and enhanced chemiluminescence. As a loading control, the expression of β-actin was detected. (B) THP-1 cells transfected with HA-RhoH or the empty vector were analysed for CD123 expression by FACS using a CD123-APC antibody. Data are presented as percentage of the mean fluorescence signal for CD123 (mean ± SD, n = 3, *P ≤ 0.05). (C) IRF-1 mRNA expression is decreased in RhoH over-expressing THP-1 cells. Quantification of IRF-1 mRNA expression levels by quantitative SYBR-green PCR in control cells and RhoH-transfected THP-1 cells using human IRF-1 specific primers and GAPDH as a reference gene. Data were normalised to the expression in control cells and are presented as fold induction (mean ± SD, n = 3; ***P ≤ 0.005).
Figure 5 The AML cell line THP-1 displays an siRhoH phenotype. (A) THP-1 cells were either transfected with HA-RhoH or the empty vector, lysed and separated by SDS-PAGE. Detection of HA-RhoH was performed with an HA-specific antibody and enhanced chemiluminescence. As a loading control, the expression of β-actin was detected. (B) THP-1 cells transfected with HA-RhoH or the empty vector were analysed for CD123 expression by FACS using a CD123-APC antibody. Data are presented as percentage of the mean fluorescence signal for CD123 (mean ± SD, n = 3, *P ≤ 0.05). (C) IRF-1 mRNA expression is decreased in RhoH over-expressing THP-1 cells. Quantification of IRF-1 mRNA expression levels by quantitative SYBR-green PCR in control cells and RhoH-transfected THP-1 cells using human IRF-1 specific primers and GAPDH as a reference gene. Data were normalised to the expression in control cells and are presented as fold induction (mean ± SD, n = 3; ***P ≤ 0.005).CC BY · PubMed Central

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Metadata

Authors
Gündogdu, M. S., Liu, H., Metzdorf, D., Hildebrand, D., Aigner, M., Aktories, K., Heeg, K., & Kubatzky, K. F.
Affiliations
  • Heidelberg University (DE)
  • University of Bern (CH)
  • Universitätsklinikum Erlangen (DE)
  • University of Freiburg (DE)
Corresponding author
Kubatzky, Katharina F
ORCID
Venue
Molecular Cancer
Publisher
Springer Science and Business Media LLC
Published
2010-08-25
Volume
9
Issue
1
Pages
225-225
ISSN
1476-4598 (ISSN-L)
DOI
10.1186/1476-4598-9-225
PMC
PMC2936343
OpenAlex
W2148472599
Semantic Scholar
82c29c1276b025e4a6c8dee1e648fcc5734f17cf
Cited by
29
References
38
Citations by year
2025: 2 · 2024: 4 · 2023: 4 · 2022: 3 · 2021: 1 · 2020: 1 · 2018: 1 · 2016: 2 · 2015: 1 · 2014: 3 · 2013: 1 · 2012: 3
FWCI
0.90
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Molecular Cancer (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • heiDOK (Heidelberg University) (acceptedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
Deutsche Forschungsgemeinschaft
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Cytokine Signaling Pathways and Interactions
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
Keywords
RhoH, Rho GTPases, STAT5, STAT1, CD123, JAK/STAT signaling, acute myeloid leukemia, p21, p27, cell proliferation, hematopoietic cells, Ba/F3 cells, THP-1 cells
MeSH terms
Animals, Cell Line, Interleukin-3, Transcription Factors, Signal Transduction, Up-Regulation, Receptors, Interleukin-3, rho GTP-Binding Proteins, Cell Proliferation, Cyclin-Dependent Kinase Inhibitor p21, Cyclin-Dependent Kinase Inhibitor p27, STAT Transcription Factors, Mice

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CoChat. (2026). Citation check: The haematopoietic GTPase RhoH modulates IL3 signalling through regulation of STAT activity and IL3 receptor expression. https://app.cochat.ai/papers/10.1186/1476-4598-9-225
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Gündogdu, M. S., Liu, H., Metzdorf, D., Hildebrand, D., Aigner, M., Aktories, K., Heeg, K., & Kubatzky, K. F. (2010). The haematopoietic GTPase RhoH modulates IL3 signalling through regulation of STAT activity and IL3 receptor expression. Molecular Cancer. https://doi.org/10.1186/1476-4598-9-225