Involvement of N-terminal-extended Form of Sphingosine Kinase 2 in Serum-dependent Regulation of Cell Proliferation and Apoptosis

Abstract

Sphingosine kinase (SPHK) 1 is implicated in the regulation of cell proliferation and anti-apoptotic processes by catalyzing the formation of an important bioactive messenger, sphingosine 1-phosphate. Unlike the proliferative action of SPHK1, another isozyme, SPHK2, has been shown to possess anti-proliferative or pro-apoptotic action. Molecular mechanisms of SPHK2 action, however, are largely unknown. The present studies were undertaken to characterize the N-terminal-extended form of SPHK2 (SPHK2-L) by comparing it with the originally reported form, SPHK2-S. Real-time quantitative PCR analysis revealed that SPHK2-L mRNA is the major form in several human cell lines and tissues. From sequence analyses it was concluded that SPHK2-L is a species-specific isoform that is expressed in human but not in mouse. At the protein level it has been demonstrated by immunoprecipitation studies that SPHK2-L is the major isoform in human hepatoma HepG2 cells. SPHK2-L, when expressed in human embryonic kidney (HEK) 293 cells, did not show any inhibition of DNA synthesis in the presence of serum, whereas it showed marked inhibition in the absence of serum. Moreover, serum deprivation resulted in the translocation of SPHK2-L into the nuclei. In addition, serum deprivation induced SPHK2-L expression in HEK293 cells. Furthermore, suppression of SPHK2 by small interfering RNA treatment prevented serum deprivation- or drug-induced apoptosis in HEK293 cells. Taken together, these results indicate that a major form of SPHK2 splice variant, SPHK2-L, in human cells does not inhibit DNA synthesis and that SPHK2-L in the induced by serum deprivation in the of cell proliferation or apoptosis the cell Sphingosine kinase (SPHK) 1 is implicated in the regulation of cell proliferation and anti-apoptotic processes by catalyzing the formation of an important bioactive messenger, sphingosine 1-phosphate. Unlike the proliferative action of SPHK1, another isozyme, SPHK2, has been shown to possess anti-proliferative or pro-apoptotic action. Molecular mechanisms of SPHK2 action, however, are largely unknown. The present studies were undertaken to characterize the N-terminal-extended form of SPHK2 (SPHK2-L) by comparing it with the originally reported form, SPHK2-S. Real-time quantitative PCR analysis revealed that SPHK2-L mRNA is the major form in several human cell lines and tissues. From sequence analyses it was concluded that SPHK2-L is a species-specific isoform that is expressed in human but not in mouse. At the protein level it has been demonstrated by immunoprecipitation studies that SPHK2-L is the major isoform in human hepatoma HepG2 cells. SPHK2-L, when expressed in human embryonic kidney (HEK) 293 cells, did not show any inhibition of DNA synthesis in the presence of serum, whereas it showed marked inhibition in the absence of serum. Moreover, serum deprivation resulted in the translocation of SPHK2-L into the nuclei. In addition, serum deprivation induced SPHK2-L expression in HEK293 cells. Furthermore, suppression of SPHK2 by small interfering RNA treatment prevented serum deprivation- or drug-induced apoptosis in HEK293 cells. Taken together, these results indicate that a major form of SPHK2 splice variant, SPHK2-L, in human cells does not inhibit DNA synthesis and that SPHK2-L in the induced by serum deprivation in the of cell proliferation or apoptosis the cell The sphingosine form of reported form of interfering embryonic form of reported form of interfering embryonic has been implicated in the regulation of a of important cell and apoptosis in has of the of an and the of an of the to of apoptosis and kinase the that the of a in the regulation of of of and been and is a with an of and the of has a in of cell proliferation by the to of the cell by the to serum deprivation or treatment SPHK2 the in the and in the in a protein In addition, in the was in SPHK2 of unknown. The of SPHK2, however, has not been demonstrated that SPHK2 is a protein and DNA synthesis when in cells reported that SPHK2 apoptosis SPHK2 has been to a of an or with in cells the of SPHK2 largely present studies were undertaken to the of N-terminal-extended SPHK2 (SPHK2-L) in with the originally reported form shown that SPHK2-L is the form, in human cell lines and that SPHK2-L is in the regulation of cell proliferation and apoptosis in with and human SPHK2-L was a human by PCR with and and The PCR was into to an of human expression has been RNA was HEK293 cells the RNA human were synthesis was with 1 of RNA with PCR was by the PCR with the of an sequence The human were by and were SPHK2-L and in 1 and SPHK2-L and and in and and and was with were of and The expression of human SPHK2 mRNA was to mRNA and of an has been the of SPHK2-L was by with an the of SPHK2-L with The was by the cells were and with were that was the and cells were by SPHK2 was or and reported were and with SPHK2-L to the studies were RNA and and and were HEK293 cells were with a of cells were another with or and with in The cells were with and was SPHK2-L the in and of the human sequence revealed the of The SPHK2-L is and of by The sequence of and a of originally reported by the and the was the is to that is by of the that that to the in is a that SPHK2-L in of of of of are and a that a 1 in SPHK2-L and that the the reported SPHK2 and the the of and SPHK2-S. The sequence is the The N-terminal-extended of 1 and are shown in and The SPHK2-L and are or The and is the the of splice in human cell lines by quantitative PCR of In 1 the was 1 and the that the mRNA SPHK2-L whereas in the was and the to mRNA SPHK2-L and SPHK2-S. that the of SPHK2-L to mRNA is the of the of PCR with these is to in human and cell lines the of PCR with 1 is to that with the that SPHK2-L is the major form expressed in human and cell lines and kidney and expressed a human RNA a the SPHK2-L and SPHK2 was to that and shown in the of expression of in these tissues. a of in the was and the that the mRNA the SPHK2-L and the the results with the were not SPHK2-L the in SPHK2-L, an SPHK2-L was a the N-terminal-extended of SPHK2-L SPHK2-L, but not SPHK2 cell in a the of SPHK2-L in cells. of SPHK2-L was SPHK2-L was in the and to a small in the of cells is to that of and is SPHK2-L the in human hepatoma HepG2 cells SPHK2-L was in the and to a in the in a that is cell that SPHK2-L is a major form the protein level SPHK2 was and the human hepatoma HepG2 cells but not cells, SPHK2-L and it cell with results the sequence analyses these results indicate that SPHK2-L is a species-specific isoform that is present in human but not in the results in of SPHK2-L in human cell of that SPHK2-L and were to by analysis with or SPHK2-S. cell were with of immunoprecipitation was of SPHK2-L and HepG2 cells were and analyses were with of is is a species-specific and major isoform in human hepatoma HepG2 cells. HepG2 and cell were with or immunoprecipitation was SPHK2 HepG2 cell with was by the The SPHK2 was with of The SPHK2 and the were are of the of the with and was the was HepG2 cell with and the by with the The was by The of was by the with a of the the in an that SPHK2-L is a major form in HepG2 cells. results were when HEK293 cell were not of DNA by SPHK2-L by shown that SPHK2 and DNA synthesis was the SPHK2 DNA synthesis was in HEK293 cells or shown in was in cells with cells, is with In when was expressed the inhibition of was in comparing the cells that expressed the to a SPHK2-L has or that SPHK2-L does not inhibit DNA synthesis the presence of reported that inhibition of DNA synthesis in cells by SPHK2 is when cells are serum with SPHK2-L, DNA synthesis was in HEK293 cells SPHK2-L or serum serum was in cells SPHK2-L when with protein cells The serum inhibition of DNA synthesis by SPHK2-L was demonstrated by cell analysis that the of cells SPHK2-L was in but not in with cells protein of DNA synthesis by SPHK2-L is by serum. HEK293 cells were with or protein and 1 in with or was to cells and and the of cells was are of to the that serum the inhibition of DNA synthesis by SPHK2-L to the regulation of of SPHK2-L by serum. HEK293 cells were with and was or serum serum SPHK2-L to in the with a small in the and the serum the of cells with was and the regulation of of SPHK2-L by serum. of serum of SPHK2-L in the was in human regulation of of and HEK293 cells were with and 1 with serum or serum were and analyses in and cells SPHK2-L were into SPHK2-L cells with cells with and and cells with were with a of cells. The are of in SPHK2-L in HEK293 the of studies the regulation of of by serum in HEK293 cells, that the of SPHK2 to when cells were in serum to SPHK2 serum HEK293 cells were in a or serum and SPHK2 or mRNA by quantitative PCR the of and SPHK2-L serum resulted in and in and SPHK2 mRNA results were when quantitative PCR was a 1 that is to SPHK2-L, that serum deprivation SPHK2-L expression but not SPHK2-S. the of HEK293 cells in serum however, the of and did not with the cells in serum SPHK2 SPHK2 in a of SPHK2 by HEK293 or the of SPHK2 induced by serum in HEK293 cells the level of SPHK2 was by with treatment with the SPHK2 mRNA level in HEK293 cells HEK293 cells apoptosis serum deprivation in cells and serum apoptosis was by treatment and by suppression of SPHK2 expression was serum cell serum resulted in a of cell to of cells, in cells not treatment the cells results indicate that SPHK2 in the regulation of serum deprivation- or drug-induced apoptosis by suppression of SPHK2 expression HEK293 cells were with SPHK2 or SPHK2 and with RNA was the cells, and by quantitative PCR shown in 1 cells were a with or to cells with were with 1 to were and with and of apoptosis were and expressed a of cells. The results are of in are and of HEK293 cells with or SPHK2 with indicate the cells and demonstrated the of an N-terminal-extended form of SPHK2 (SPHK2-L) in human cell lines and and it a species-specific that is the major in human but that not in by several a the N-terminal-extended of SPHK2-L, with SPHK2-L but not with and and HepG2 but not cell of the SPHK2 protein an was by an the of mRNA by 1 1 and is to SPHK2-L, is the that by and is to and SPHK2-L the was the not is present in the human cell lines to the of an or that protein or and in the present studies the these the not of the SPHK2-L in a not SPHK2-L when expressed in cells and by an showed a but not a it demonstrated in the and the when by an SPHK2-L in the and the when by or not results that SPHK2-L in a with in the and with the of the by SPHK2-L present in SPHK2-L expressed in cells showed in not the of the of of SPHK2-L the cell In cells it was in the whereas it was in the and the in HepG2 cells the it was in the of HEK293 cells of SPHK2-L was not the cell but of a the of of reported that DNA synthesis and cell when expressed in cells In the present studies demonstrated that SPHK2-L has a to inhibit DNA synthesis that SPHK2-L is the major form in is cells SPHK2 is in the is important to that SPHK2 possess the to inhibit cell proliferation and apoptosis serum suppression of SPHK2 expression by prevented serum apoptosis The of SPHK2 to inhibit cell proliferation is that of SPHK1, cell and demonstrated that to SPHK1, the to by of with the SPHK2, in cells when was to by with a sequence the protein cell proliferation by cell Moreover, reported that translocation of to the by by kinase is of Taken together, these that of or is an important of cell with of sphingosine level by the the present results that serum SPHK2 translocation into the and expression of SPHK2 serum apoptosis SPHK2-L to to a level in the The of the Molecular of action in the to The sphingosine form of reported form of interfering embryonic form of reported form of interfering embryonic has been implicated in the regulation of a of important cell and apoptosis in has of the of an and the of an of the to of apoptosis and sphingosine sphingosine kinase human SPHK2 N-terminal-extended form of SPHK2 originally reported form of SPHK2 serum small interfering RNA human embryonic kidney sphingosine sphingosine kinase human SPHK2 N-terminal-extended form of SPHK2 originally reported form of SPHK2 serum small interfering RNA human embryonic kidney Sphingosine kinase the that the of a in the regulation of of of and been and is a with an of and the of has a in of cell proliferation by the to of the cell by the to serum deprivation or treatment SPHK2 the in the and in the in a protein In addition, in the was in SPHK2 of unknown. The of SPHK2, however, has not been demonstrated that SPHK2 is a protein and DNA synthesis when in cells reported that SPHK2 apoptosis SPHK2 has been to a of an or with in cells the of SPHK2 largely unknown. The present studies were undertaken to the of N-terminal-extended SPHK2 (SPHK2-L) in with the originally reported form shown that SPHK2-L is the form, in human cell lines and that SPHK2-L is in the regulation of cell proliferation and apoptosis in with serum. and human SPHK2-L was a human by PCR with and and The PCR was into to an of human expression has been RNA was HEK293 cells the RNA human were synthesis was with 1 of RNA with PCR was by the PCR with the of an sequence The human were by and were SPHK2-L and in 1 and SPHK2-L and and in and and and was with were of and The expression of human SPHK2 mRNA was to mRNA and of an has been the of SPHK2-L was by with an the of SPHK2-L with The was by the cells were and with were that was the and cells were by SPHK2 was or and reported were and with SPHK2-L to the studies were RNA and and and were HEK293 cells were with a of cells were another with or and with in The cells were with and was and human SPHK2-L was a human by PCR with and and The PCR was into to an of human expression has been Real-time RNA was HEK293 cells the RNA human were synthesis was with 1 of RNA with PCR was by the PCR with the of an sequence The human were by and were SPHK2-L and in 1 and SPHK2-L and and in and and and was with were of and The expression of human SPHK2 mRNA was to mRNA and of an has been the of SPHK2-L was by with an the of SPHK2-L with The was by the HEK293 cells were and with were that was the and cells were by SPHK2 was or and reported were and with SPHK2-L to the studies were RNA and and and were HEK293 cells were with a of cells were another with or and with in The cells were with and was SPHK2-L the in and of the human sequence revealed the of The SPHK2-L is and of by The sequence of and a of originally reported by the and the was the is to that is by of the that that to the in is a that SPHK2-L in the of splice in human cell lines by quantitative PCR of In 1 the was 1 and the that the mRNA SPHK2-L whereas in the was and the to mRNA SPHK2-L and SPHK2-S. that the of SPHK2-L to mRNA is the of the of PCR with these is to in human and cell lines the of PCR with 1 is to that with the that SPHK2-L is the major form expressed in human and cell lines and kidney and expressed a human RNA a the SPHK2-L and SPHK2 was to that and shown in the of expression of in these tissues. a of in the was and the that the mRNA the SPHK2-L and the the results with the were not SPHK2-L the in SPHK2-L, an SPHK2-L was a the N-terminal-extended of SPHK2-L SPHK2-L, but not SPHK2 cell in a the of SPHK2-L in cells. of SPHK2-L was SPHK2-L was in the and to a small in the of cells is to that of and is SPHK2-L the in human hepatoma HepG2 cells SPHK2-L was in the and to a in the in a that is cell that SPHK2-L is a major form the protein level SPHK2 was and the human hepatoma HepG2 cells but not cells, SPHK2-L and it cell with results the sequence analyses these results indicate that SPHK2-L is a species-specific isoform that is present in human but not in the results in of SPHK2-L in human cell of that SPHK2-L and were to by analysis with or SPHK2-S. cell were with of immunoprecipitation was of SPHK2-L and HepG2 cells were and analyses were with of is is a species-specific and major isoform in human hepatoma HepG2 cells. HepG2 and cell were with or immunoprecipitation was SPHK2 HepG2 cell with was by the The SPHK2 was with of The SPHK2 and the were are of the of the with and was the was HepG2 cell with and the by with the The was by The of was by the with a of the the in an that SPHK2-L is a major form in HepG2 cells. results were when HEK293 cell were not of DNA by SPHK2-L by shown that SPHK2 and DNA synthesis was the SPHK2 DNA synthesis was in HEK293 cells or shown in was in cells with cells, is with In when was expressed the inhibition of was in comparing the cells that expressed the to a SPHK2-L has or that SPHK2-L does not inhibit DNA synthesis the presence of reported that inhibition of DNA synthesis in cells by SPHK2 is when cells are serum with SPHK2-L, DNA synthesis was in HEK293 cells SPHK2-L or serum serum was in cells SPHK2-L when with protein cells The serum inhibition of DNA synthesis by SPHK2-L was demonstrated by cell analysis that the of cells SPHK2-L was in but not in with cells protein of DNA synthesis by SPHK2-L is by serum. HEK293 cells were with or protein and 1 in with or was to cells and and the of cells was are of to the that serum the inhibition of DNA synthesis by SPHK2-L to the regulation of of SPHK2-L by serum. HEK293 cells were with and was or serum serum SPHK2-L to in the with a small in the and the serum the of cells with was and the regulation of of SPHK2-L by serum. of serum of SPHK2-L in the was in human regulation of of and HEK293 cells were with and 1 with serum or serum were and analyses in and cells SPHK2-L were into SPHK2-L cells with cells with and and cells with were with a of cells. The are of in SPHK2-L in HEK293 the of studies the regulation of of by serum in HEK293 cells, that the of SPHK2 to when cells were in serum to SPHK2 serum HEK293 cells were in a or serum and SPHK2 or mRNA by quantitative PCR the of and SPHK2-L serum resulted in and in and SPHK2 mRNA results were when quantitative PCR was a 1 that is to SPHK2-L, that serum deprivation SPHK2-L expression but not SPHK2-S. the of HEK293 cells in serum however, the of and did not with the cells in serum SPHK2 SPHK2 in a of SPHK2 by HEK293 or the of SPHK2 induced by serum in HEK293 cells the level of SPHK2 was by with treatment with the SPHK2 mRNA level in HEK293 cells HEK293 cells apoptosis serum deprivation in cells and serum apoptosis was by treatment and by suppression of SPHK2 expression was serum cell serum resulted in a of cell to of cells, in cells not treatment the cells results indicate that SPHK2 in the regulation of serum deprivation- or drug-induced apoptosis by suppression of SPHK2 expression HEK293 cells were with SPHK2 or SPHK2 and with RNA was the cells, and by quantitative PCR shown in 1 cells were a with or to cells with were with 1 to were and with and of apoptosis were and expressed a of cells. The results are of in are and of HEK293 cells with or SPHK2 with indicate the cells and mRNA SPHK2-L the in and of the human sequence revealed the of The SPHK2-L is and of by The sequence of and a of originally reported by the and the was the is to that is by of the that that to the in is a that SPHK2-L in mouse. the of splice in human cell lines by quantitative PCR of In 1 the was 1 and the that the mRNA SPHK2-L whereas in the was and the to mRNA SPHK2-L and SPHK2-S. that the of SPHK2-L to mRNA is the of the of PCR with these is to in human and cell lines the of PCR with 1 is to that with the that SPHK2-L is the major form expressed in human and cell lines and kidney and expressed a human RNA a the SPHK2-L and SPHK2 was to that and shown in the of expression of in these tissues. a of in the was and the that the mRNA the SPHK2-L and the the results with the were not SPHK2-L the in SPHK2-L, an SPHK2-L was a the N-terminal-extended of SPHK2-L SPHK2-L, but not SPHK2 cell in a the of SPHK2-L in cells. of SPHK2-L was SPHK2-L was in the and to a small in the of cells is to that of and is SPHK2-L the in human hepatoma HepG2 cells SPHK2-L was in the and to a in the in a that is cell that SPHK2-L is a major form the protein level SPHK2 was and the human hepatoma HepG2 cells but not cells, SPHK2-L and it cell with results the sequence analyses these results indicate that SPHK2-L is a species-specific isoform that is present in human but not in the results in the of the with and was the was HepG2 cell with and the by with the The was by The of was by the with a of the the in an that SPHK2-L is a major form in HepG2 cells. results were when HEK293 cell were not of DNA by SPHK2-L by shown that SPHK2 and DNA synthesis was the SPHK2 DNA synthesis was in HEK293 cells or shown in was in cells with cells, is with In when was expressed the inhibition of was in comparing the cells that expressed the to a SPHK2-L has or that SPHK2-L does not inhibit DNA synthesis the presence of reported that inhibition of DNA synthesis in cells by SPHK2 is when cells are serum with SPHK2-L, DNA synthesis was in HEK293 cells SPHK2-L or serum serum was in cells SPHK2-L when with protein cells The serum inhibition of DNA synthesis by SPHK2-L was demonstrated by cell analysis that the of cells SPHK2-L was in but not in with cells protein SPHK2-L to the that serum the inhibition of DNA synthesis by SPHK2-L to the regulation of of SPHK2-L by serum. HEK293 cells were with and was or serum serum SPHK2-L to in the with a small in the and the serum the of cells with was and the regulation of of SPHK2-L by serum. of serum of SPHK2-L in the was in human in SPHK2-L in HEK293 the of studies the regulation of of by serum in HEK293 cells, that the of SPHK2 to when cells were in serum to SPHK2 serum HEK293 cells were in a or serum and SPHK2 or mRNA by quantitative PCR the of and SPHK2-L serum resulted in and in and SPHK2 mRNA results were when quantitative PCR was a 1 that is to SPHK2-L, that serum deprivation SPHK2-L expression but not SPHK2-S. the of HEK293 cells in serum however, the of and did not with the cells in serum of SPHK2 by HEK293 or the of SPHK2 induced by serum in HEK293 cells the level of SPHK2 was by with treatment with the SPHK2 mRNA level in HEK293 cells HEK293 cells apoptosis serum deprivation in cells and serum apoptosis was by treatment and by suppression of SPHK2 expression was serum cell serum resulted in a of cell to of cells, in cells not treatment the cells results indicate that SPHK2 in the regulation of demonstrated the of an N-terminal-extended form of SPHK2 (SPHK2-L) in human cell lines and and it a species-specific that is the major in human but that not in by several a the N-terminal-extended of SPHK2-L, with SPHK2-L but not with and and HepG2 but not cell of the SPHK2 protein an was by an the of mRNA by 1 1 and is to SPHK2-L, is the that by and is to and SPHK2-L the was the not is present in the human cell lines to the of an or that protein or and in the present studies the these the not of the SPHK2-L in a not SPHK2-L when expressed in cells and by an showed a but not a it demonstrated in the and the when by an SPHK2-L in the and the when by or not results that SPHK2-L in a with in the and with the of the by SPHK2-L present in SPHK2-L expressed in cells showed in not the of the of of SPHK2-L the cell In cells it was in the whereas it was in the and the in HepG2 cells the it was in the of HEK293 cells of SPHK2-L was not the cell but of a the of of reported that DNA synthesis and cell when expressed in cells In the present studies demonstrated that SPHK2-L has a to inhibit DNA synthesis that SPHK2-L is the major form in is cells SPHK2 is in the is important to that SPHK2 possess the to inhibit cell proliferation and apoptosis serum suppression of SPHK2 expression by prevented serum apoptosis The of SPHK2 to inhibit cell proliferation is that of SPHK1, cell and demonstrated that to SPHK1, the to by of with the SPHK2, in cells when was to by with a sequence the protein cell proliferation by cell Moreover, reported that translocation of to the by by kinase is of Taken together, these that of or is an important of cell with of sphingosine level by the the present results that serum SPHK2 translocation into the and expression of SPHK2 serum apoptosis SPHK2-L to to a level in the The of the Molecular of action in the to demonstrated the of an N-terminal-extended form of SPHK2 (SPHK2-L) in human cell lines and and it a species-specific that is the major in human but that not in by several a the N-terminal-extended of SPHK2-L, with SPHK2-L but not with and and HepG2 but not cell of the SPHK2 protein an was by an the of mRNA by 1 1 and is to SPHK2-L, is the that by and is to and SPHK2-L the was the not is present in the human cell lines to the of an or that protein or and in the present studies the these the not of the SPHK2-L in a not SPHK2-L when expressed in cells and by an showed a but not a it demonstrated in the and the when by an SPHK2-L in the and the when by or not results that SPHK2-L in a with in the and with the of the by SPHK2-L present in SPHK2-L expressed in cells showed in not the of the of The of SPHK2-L the cell In cells it was in the whereas it was in the and the in HepG2 cells the it was in the of HEK293 cells of SPHK2-L was not the cell but of a the of of reported that DNA synthesis and cell when expressed in cells In the present studies demonstrated that SPHK2-L has a to inhibit DNA synthesis that SPHK2-L is the major form in is cells SPHK2 is in the is important to that SPHK2 possess the to inhibit cell proliferation and apoptosis serum suppression of SPHK2 expression by prevented serum apoptosis The of SPHK2 to inhibit cell proliferation is that of SPHK1, cell and demonstrated that to SPHK1, the to by of with the SPHK2, in cells when was to by with a sequence the protein cell proliferation by cell Moreover, reported that translocation of to the by by kinase is of Taken together, these that of or is an important of cell with of sphingosine level by the the present results that serum SPHK2 translocation into the and expression of SPHK2 serum apoptosis SPHK2-L to to a level in the The of the Molecular of action in the to with with