Elsevier

Leukemia Research

Volume 28, Issue 1, January 2004, Pages 71-82
Leukemia Research

Down-regulation of SHP1 and up-regulation of negative regulators of JAK/STAT signaling in HTLV-1 transformed cell lines and freshly transformed human peripheral blood CD4+ T-Cells

https://doi.org/10.1016/S0145-2126(03)00158-9Get rights and content

Abstract

Adult T-cell leukemia (ATL) is an aggressive malignancy that is associated with human T-cell lymphotropic virus I (HTLV-I) infection. HTLV-I transformed T-cell lines and fresh ATL cells are characterized by constitutive activation of the interleukin-2 receptor (IL-2R) signaling pathway however, the mechanism(s) responsible for constitutive IL-2R activation are unknown. To further examine the cause of this signaling pathway deregulation, we measured mRNA and protein expression levels by real-time PCR and Western blots, respectively, of four negative regulators of the IL-2R signaling pathway including src homology 2 (SH2)-containing phosphatase (SHP1), cytokine-inducible (CIS) SH2-containing protein, suppressor of cytokine signaling-1 (SOCS1) and protein inhibitor of activated signal transducer and activator of transcription 3 (STAT3) (PIAS3) in six HTLV-1 negative and seven HTLV-1 positive T-cell leukemia lines. The activation status of the JAK/STAT pathway was also examined. SHP1 mRNA and protein expression levels were selectively down regulated in all HTLV-1-infected transformed cell lines, while CIS, SOCS1, and PIAS3 protein expression were markedly but variably upregulated and the cells showed evidence of constitutive STAT3 activation. In acutely HTLV-1 infected primary CD4+ T-cells there was a gradual loss of SHP1 expression over 10 weeks in culture which correlated with progression from immortalization to transformation and loss of IL-2 dependence for growth. Two transformed cell lines that were established following HTLV-1 infection showed loss of SHP1 expression and overexpression of CIS, SOCS1, PIAS3. However, this overexpression was not adequate to block constitutive activation of the JAK/STAT pathway. Thus, multiple levels of IL-2 receptor signal deregulation are found in HTLV-1 transformed cells, which may be a result of early loss of SHP1 expression.

Introduction

Human T-cell lymphotropic virus I (HTLV-I) is the etiological agent for adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [1]. Deregulation of IL-2 receptor (IL-2R) signaling appears to play an important role in the events that lead to immortalization and oncogenic transformation of CD4+ T-cells by HTLV-1. HTLV-1-infected peripheral blood T-cells exhibit an initial phase of interleukin-2 (IL-2)-dependent growth; over time, by an unknown mechanism the cells become IL-2 independent [2]. Typically the transformed cell lines display activation of the JAK/signal transducer and activator of transcription (STAT) signaling pathway with increased constitutive phosphorylation of the IL-2R complex, including IL-2Rβ, the Janus kinases JAK1 and JAK3 and the signal transducers and activators of transcription, STAT3 and STAT5 [2], [3], [4].

Many studies have focused on the mechanisms by which IL-2 exerts its actions and how activation of the IL-2R is controlled [5]. Recently, several control mechanisms for the negative regulation of cytokine signaling have been elucidated. These include protein degradation [6], [7], [8], the activation of phosphatases such as SHP1 [6], [9], a recently discovered family of small src-homology 2 (SH2)-containing proteins including cytokine-inducible (CIS SH2-containing protein) [10], and seven SOCS proteins (SOCS1–7), suppressor of cytokine signaling [11], [12], [13], [14], and protein inhibitor of activated STAT (PIAS) proteins including PIAS1 and PIAS3 [15], [16]. Each of these negative regulators has a distinct mode of action [17], [18]. For example, SHP1 is present constitutively in cells, and as such is able to downregulate signaling immediately when receptor/kinase complexes are activated. In particular, IL-2 induces association of SHP1 with the IL-2R complex, and once SHP1 is recruited to the activated receptor it is able to decrease tyrosine phosphorylation of IL-2Rβ and the associated tyrosine kinases JAK1 and JAK3 [19]. CIS is induced in hematopoietic cells within 30 min of stimulation by IL-2 and other cytokines [10], [20]. CIS interacts with the A region of IL-2Rβ which also mediates the association of IL-2Rβ with Lck and JAK3 and inhibits functions associated with both of these kinases: Lck-mediated phosphorylation of IL-2Rβ and IL-2-mediated JAK3 activation and subsequent activation of STAT5 [20]. Like CIS, SOCS1 is also strongly induced by IL-2, can associate with IL-2Rβ through the A region of IL-2Rβ and can potently inhibit IL-2-induced STAT5 function [21]. However, the association of SOCS1 with IL-2Rβ is not essential for its inhibitory activity. More likely, it is the direct binding of SOCS1 with both JAK1 and JAK3 and the resulting inhibition of JAK1 tyrosine phosphorylation and kinase activity that is responsible for its negative regulatory effects [21]. SOCS3 is also strongly induced and in parallel phosphorylated by IL-2 stimulation, is associated with JAK1 and can inhibit JAK1 phosphorylation and kinase activity and markedly reduce IL-2-induced tyrosine phosphorylation of STAT5b [22]. PIAS3 can inhibit IL-6 induced STAT3-dependent transcriptional activation however, the role of PIAS3 in IL-2 signaling has not been reported. The fact that PIAS3 appears to be constitutively expressed suggests that the PIAS proteins may act as a basal-level barrier to STAT protein action, rather than as cytokine-inducible negative feedback effectors [18]. Finally, the deubiquitinating enzyme DUB-2, which is induced in response to IL-2 has been reported to markedly prolong IL-2-induced STAT5 phosphorylation even after growth factor withdraw [8].

In two recent reports, SHP1 expression was found to be greatly decreased or undetectable in several IL-2-independent HTLV-1 transformed T-cell lines that exhibit constitutive JAK/STAT activation [19], [23]. In addition, in HTLV-1 infected T-cells, down-regulation of SHP1 was also found to correlate with the acquisition of IL-2 independence. However, these reports did not evaluate the expression levels of other negative regulators of cytokine signaling or the expression levels of SHP1 at early times following HTLV-1-infection of CD4+ T-cells. We therefore undertook this study to evaluate the levels of mRNA and protein expression of several negative regulators of IL-2 signaling in acutely and chronically HTLV-1-infected transformed cell lines as well as the activation status of the JAK/STAT signaling pathway. We observed that not only were SHP1 levels low to undetectable but the levels of inducible (CIS and SOCS1) and constitutive (PIAS3) negative regulators were markedly but variably elevated. We also found that there was constitutive activation of the JAK/STAT pathway despite overexpression of these downstream negative regulators. Furthermore, a time course of HTLV-1 infection of human peripheral blood CD4+ T-cells established that the loss of SHP1 expression, acquisition of IL-2 independence, overexpression of CIS, SOCS1 and PIAS3 and constitutive activation of the JAK/STAT pathway occur early in the transformation process.

Section snippets

Cell lines and culture conditions

The six HTLV-1 negative cell lines (obtained from ATCC) used in this study are the following: HUT78—human cutaneous T-cell lymphoma cell line [24]; SupT1—non-Hodgkin’s T-cell lymphoma line [25]; Molt4 Clone8—acute lymphoblastic leukemia line [26]; CEM-T4—human T-lymphoblastoid line [27]; Jurkat Clone E6-1 [28]; COS-7—African green monkey kidney fibroblast-like cell line transformed with SV40 [29]. The seven HTLV-1 positive cell lines included: HUT102—[30]; MT2—in vitro transformed and

Analyses of mRNA and protein expression levels of negative regulators of JAK/STAT pathway in HTLV-1 negative and positive cell lines

Quantitative real-time RT-PCR and Western blot analyses were used to assess the mRNA and protein expression levels of several negative regulators of the JAK/STAT signaling pathway, respectively. In total, 12 T-cell leukemia cell lines were evaluated; five were HTLV-1 negative (HUT78, SupT1, Molt4 Clone8, CEM-T4, Jurkat E6-1) and seven were HTLV-1 positive cell lines (MT2, MT4, C8166-45, OS-P2, C10/MJ and SP). Of the latter, only the MT2 and OSP2 cell lines produce infectious HTLV-1 viruses [31]

Discussion

The results of this study demonstrate that SHP1 expression is uniformly downregulated in HTLV-1 transformed cell lines at both the mRNA and protein level compared to non-HTLV-1 transformed leukemia T-cell lines. In addition, variable patterns of upregulation of downstream inducible (CIS and SOCS1) and constitutive (PIAS3) negative regulators of JAK/STAT signaling were also observed. However, the increase in CIS, SOCS1 and PIAS3 expression was not sufficient to overcome loss of signal regulation

Acknowledgements

This work was supported by the National Foundation for Cancer Research (W.A.M.), and by a joint Dana-Farber Cancer Institute-Beth-Israel Deaconess Medical Center and Children’s Hospital Center for AIDS Research (CFAR) Grant. The authors also wish to acknowledge the NIH AIDS Research and Reference Reagent Program for their invaluable supply of cell lines and other materials for this project.

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