Background Chronic activation of the Janus kinase (JAK)/ Signal Transducer and Activator of Transcription (STAT) signaling pathways is a hallmark of a variety of B cell lymphomas including the classical Hodgkin’s lymphoma (cHL). Constitutive JAK/STAT signaling is crucial for survival and proliferation of Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of cHL. Although the exact molecular basis of this constitutive JAK/STAT signaling in cHL has not been understood completely, accumulating reports highlight the role of an inactivation or reduced expression of negative JAK/STAT regulators like Silencer of Cell signaling 1 (SOCS1) or Protein-Tyrosine Phosphatase 1B (PTP1B) in this process. Given the essential role of PTP1B and SOCS1 for the constitutive JAK-STAT signaling in cHL, the identification and characterization of the regulatory mechanisms controlling the activity of these negative JAK/STAT regulators is crucial for the understanding of their role for the pathogenesis of cHL. Here, we report the expression of different truncated PTP1B mRNA variants identified in cHL cell lines and primary cHL tumor samples lacking either one or several exon sequences. We describe the expression of a shorter PTP1B isoform in cHL as a novel negative control mechanism for PTP1B involved in the dis-regulation of the JAK-STAT pathway.
Methods PTP1B mRNA of cHL cases and cell lines was isolated and sequenced by Sanger sequencing. PTP1B protein expression was determined by western blot and immunohistochemistry analyses. Expression analysis of PTP1B mRNA variants was done by qPCR. To determine the role of PTP1BΔ6 for JAK/STAT signaling, stably transfected L428 cell clones either expressing PTP1BWT, PTP1BC215S, or PTP1BΔ6 were generated. Functional analysis of the novel PTP1BΔ6 variant was achieved by western blot analysis using phospho-STAT antibodies, luciferase reporter analysis and EMSA. The impact of the PTP1B variants on cell proliferation was examined by MTS assays. Results In order to determine the molecular basis of the reduced or absent PTP1B expression in selected cHL cases, we isolated and sequenced the PTP1B mRNA. In contrast to the previously published study highlighting inactivating point mutations in the PTP1B gene of PMBL and cHL cases, we obtained a set of PTP1B variants which lack different exon sequences. One of these novel PTP1B variants, lacking exon 6 (PTP1BΔ6), was found to be expressed at low levels in cHL cell lines. However, serum stimulation of cHL augmented the expression of PTP1BΔ6 significantly. Functional characterization of PTP1BΔ6 revealed a positive impact on IFNγ and IL-4 induced JAK/STAT activity in HEK293 or HEK293-STAT6 cells, and on the basal STAT activity in stably transfected L-428 and U-HO1 cHL cell lines. Furthermore, PTP1BΔ6 expression increased the proliferation of L-428 and U-HO1 cells and the reduced cytotoxic effect of the chemotherapeutical agents gemcitabine and etoposide distinctively.
Conclusions Collectively, these data suggest that the activity of PTP1B in cHL is regulated by different molecular mechanisms including the expression of shorter PTP1B variants like the novel PTP1BΔ6 variant which acts as a positive regulator of JAK/STAT signaling in cHL.