Table 1.
Sample Category Statistics.
Table 2.
Differential expression of genes in the JAK–STAT signaling pathway in MPN patients.
Figure 1.
Expression of SOCS3 and SOCS1 mRNA in MPN patients.
A. SOCS3 mRNA levels in peripheral blood of MPN patients determined by qPCR (not PCR array) were calibrated with quantities of HPRT1 mRNA and plotted against JAK2 V617F mutation burden. The values are represented with an arbitrary unit. The line and p value for the slope were calculated based on a linear regression model. B. SOCS1 mRNA levels plotted as in A. C. SOCS3 mRNA levels were plotted by disease category. Control represents the healthy volunteers, and ET− and ET+ represent V617F-negative and -positive ET patients, respectively. PV represents PV patients. The p values were calculated by a t-test. D. SOCS1 mRNA levels plotted as in C.
Figure 2.
Expression of SPI1 mRNA in MPN patients.
A. SPI1 mRNA levels in peripheral blood of MPN patients determined by qPCR (not PCR array) were calibrated with quantities of HPRT1 mRNA and plotted against JAK2 V617F mutation burden. The values are represented with an arbitrary unit. The line and p value for the slope were calculated based on a linear regression model. B. SPI1 mRNA levels were plotted by disease category. Control represents the healthy volunteers, and ET− and ET+ represent V617F-negative and -positive ET patients, respectively. PV represents PV patients. The p values were calculated by a t-test.
Figure 3.
Induction of SPI1 mRNA in K562 cells overexpressing V617F-type JAK2.
A. Western blots showing the amounts of JAK2 protein inK562 and HEL cells infected with retrovirus vector encoding either wild-type (JAK2 WT) or V617F-type JAK2 (JAK2 V617F) or a mock vector (vector) and maintained in the presence of puromycin. The intensities of bands were calibrated with the band intensities of elongation factor 2 (EF-2) protein. Fold over-expression is shown below as the value for the mock infectant as 1. The results of two independent infections are shown under experiments (exp.) 1 and 2. Alexa 680-labeled secondary antibodies were used. B. SPI1 mRNA levels in K562 cells prepared by the retroviral infection shown in A, along with those in non-infected K562 and HEL cells. The results were calibrated with 18S ribosomal RNA amount and represented with an arbitrary unit.
Figure 4.
Reduction of SOCS3 and SPI1 mRNA in HEL cells transfected with JAK2 siRNAs.
A. Western blots of JAK2 protein in HEL and K562 cells treated with siRNAs against JAK2 are shown. EF-2 was detected as a loading control. Cell lysates were prepared 24 h after siRNA transfection. Proteins derived from 1×105 cells were loaded onto each lane. Alexa 680-labeled secondary antibodies were used. Three types of siRNA against JAK2 (siRNA1–3) and a negative control siRNA (NC1) are described in the Materials and Methods section. Fold change represents a ratio of band intensity of JAK2 and that of EF-2. B. SOCS3 mRNA amount determined by qPCR. RNA was prepared 48 h after siRNA transfection. The values are expressed with an arbitrary unit as the mean of NC1 and NC2-treated HEL cells as 1. Error bars represent standard errors for triplicate measurements. C. SPI1 mRNA amount shown as in B.
Figure 5.
Effect of pharmacological inhibition of JAK2 and ABL1 kinases on SOCS3 and SPI1 expression.
A. Western blots of total JAK2 and phosphorylated JAK2 (P-JAK2) are shown. EF-2 was detected as a loading control. Cell lysates were prepared 24 h after addition of indicated concentrations of AG490 to the culture medium. Proteins derived from 5×105 cells were loaded onto each lane. Horseradish peroxidase-labeled secondary antibodies were used. B. SOCS3 and SPI1 mRNA amount determined by qPCR in cells 24 h after addition of indicated concentrations of AG490. The values are expressed with an arbitrary unit. Data at 48 h were similar (not shown). C. SOCS3 and SPI1 mRNA amount determined by qPCR in cells 48 h after addition of indicated concentrations of imatinib. The values are expressed with an arbitrary unit.
Figure 6.
Proposed signaling pathways leading to SPI1 and SOCS3 gene expression.
A thick arrow toward hematopoietic transcription factor PU.1 encoded by SPI1 gene is a novel pathway reported in this study. Signaling from ABL1 to STAT5A, STAT5B, or STAT3 (dotted arrow) leading to SOCS3 and PU.1 expression is suggested by this study but its generalization in leukemic patients requires further validation. A negative feedback loop from JAK2 to STAT proteins and to SOCS3, which inhibits JAK2, was previously established. Enhanced expression of PU.1 may be involved in MPN development via its transcriptional control of genes regulating cellular proliferation and differentiation.