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MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

Xu H, Yan Y, Williams MS, Carey GB, Yang J, Li H, Zhang GX, Rostami A - PLoS ONE (2010)

Bottom Line: CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively.In contrast, knockdown of MS4a4B accelerated T cell proliferation.MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America. hui.xu@jefferson.edu

ABSTRACT
MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

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Expression of cell cycle-regulatory genes in MS4a4B-LV-infected EL4 cells.A, Total RNA was isolated from MS4a4B-LV vector- or mock LV vector-infected EL4 cells. Expression of 84 key regulatory genes on cell cycle progression was determined with real time PCR array analysis kit (Cat#: PAMM-020, SABiosciences, Gaithersburg, MD). Data were analyzed with on-line analysis software according to the manufacturer's instructions and presented as scatter plot with fold change of genes. Red dot: genes increased by ≥4 fold; blue dot: genes decreased by ≥4 fold. B, Genes with fold change ≥4 were shown as column figure. C, MS4a4B (or control) vector-infected EL4 cells were synchronized and stimulated with 15% FCS for 8 hours. Cell lysates (100 μg) were immunoprecipitated with anti-Cdk2-protein A beads. Cdk2-cyclin activity was assayed as described in “Methods”. The same samples (25 μg) were used for western blot with anti-β-actin antibody to ensure identical loading. Relative intensity (sample vs. LV control) is shown in the lower panel. *, p<0.05. D, Cell lysates described in “C” were separated on 10% SDS gel followed by western blotting with appropriate antibodies as indicated. E. Relative intensity of blots in “D” (sample vs. internal control (β-actin)) was determined by densitometry. *, p<0.05.
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pone-0013780-g005: Expression of cell cycle-regulatory genes in MS4a4B-LV-infected EL4 cells.A, Total RNA was isolated from MS4a4B-LV vector- or mock LV vector-infected EL4 cells. Expression of 84 key regulatory genes on cell cycle progression was determined with real time PCR array analysis kit (Cat#: PAMM-020, SABiosciences, Gaithersburg, MD). Data were analyzed with on-line analysis software according to the manufacturer's instructions and presented as scatter plot with fold change of genes. Red dot: genes increased by ≥4 fold; blue dot: genes decreased by ≥4 fold. B, Genes with fold change ≥4 were shown as column figure. C, MS4a4B (or control) vector-infected EL4 cells were synchronized and stimulated with 15% FCS for 8 hours. Cell lysates (100 μg) were immunoprecipitated with anti-Cdk2-protein A beads. Cdk2-cyclin activity was assayed as described in “Methods”. The same samples (25 μg) were used for western blot with anti-β-actin antibody to ensure identical loading. Relative intensity (sample vs. LV control) is shown in the lower panel. *, p<0.05. D, Cell lysates described in “C” were separated on 10% SDS gel followed by western blotting with appropriate antibodies as indicated. E. Relative intensity of blots in “D” (sample vs. internal control (β-actin)) was determined by densitometry. *, p<0.05.

Mentions: To dissect the underlying mechanism of MS4a4B-mediated regulation of cell cycle, we analyzed gene expression that regulates cell cycle transition by real-time PCR array. We found that expression of several inhibitory genes was enhanced in MS4a4B-lentivirus-infected EL4 cells, including Ink4 family protein (p16), Cip/Kip family proteins (p21 and p27) and E2f4, the latter having been found to repress gene transcription by interfering with the binding of E2f1, E2f2 and E2f3 to promoters [36]. On the other hand, expression of positive cell cycle regulators, e.g. Cyclin A1, Cyclin B1 and E2f2, was decreased (Fig. 5A and B). Another enhanced inhibitory molecule is Apbb1 (Amyloid beta (A4) precursor protein-binding, family B, member 1), also named Fe65, which is an adaptor protein localized in the nucleus. Overexpression of Fe65 in mouse fibroblasts has been shown to block cell growth by inhibiting the activation of a key S phase gene, the thymidylate synthase (TS) gene [37]. Notably, MS4a4B expression enhanced the production of the calcium/calmodulin-dependent protein kinase (CaM kinase) IIα, also known as Camk2a, whose kinase activity is dependent upon its activation by calmodulin (CaM). CaM is one of the key proteins that transduces a signal in response to increases in intracellular Ca2+. Whether MS4a4B protein is involved in regulation of Ca2+ signaling still remains to be determined. We observed no change in p53 at RNA transcription levels, suggesting that p53 is not involved in MS4a4B-mediated inhibition of cell cycle.


MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

Xu H, Yan Y, Williams MS, Carey GB, Yang J, Li H, Zhang GX, Rostami A - PLoS ONE (2010)

Expression of cell cycle-regulatory genes in MS4a4B-LV-infected EL4 cells.A, Total RNA was isolated from MS4a4B-LV vector- or mock LV vector-infected EL4 cells. Expression of 84 key regulatory genes on cell cycle progression was determined with real time PCR array analysis kit (Cat#: PAMM-020, SABiosciences, Gaithersburg, MD). Data were analyzed with on-line analysis software according to the manufacturer's instructions and presented as scatter plot with fold change of genes. Red dot: genes increased by ≥4 fold; blue dot: genes decreased by ≥4 fold. B, Genes with fold change ≥4 were shown as column figure. C, MS4a4B (or control) vector-infected EL4 cells were synchronized and stimulated with 15% FCS for 8 hours. Cell lysates (100 μg) were immunoprecipitated with anti-Cdk2-protein A beads. Cdk2-cyclin activity was assayed as described in “Methods”. The same samples (25 μg) were used for western blot with anti-β-actin antibody to ensure identical loading. Relative intensity (sample vs. LV control) is shown in the lower panel. *, p<0.05. D, Cell lysates described in “C” were separated on 10% SDS gel followed by western blotting with appropriate antibodies as indicated. E. Relative intensity of blots in “D” (sample vs. internal control (β-actin)) was determined by densitometry. *, p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2967469&req=5

pone-0013780-g005: Expression of cell cycle-regulatory genes in MS4a4B-LV-infected EL4 cells.A, Total RNA was isolated from MS4a4B-LV vector- or mock LV vector-infected EL4 cells. Expression of 84 key regulatory genes on cell cycle progression was determined with real time PCR array analysis kit (Cat#: PAMM-020, SABiosciences, Gaithersburg, MD). Data were analyzed with on-line analysis software according to the manufacturer's instructions and presented as scatter plot with fold change of genes. Red dot: genes increased by ≥4 fold; blue dot: genes decreased by ≥4 fold. B, Genes with fold change ≥4 were shown as column figure. C, MS4a4B (or control) vector-infected EL4 cells were synchronized and stimulated with 15% FCS for 8 hours. Cell lysates (100 μg) were immunoprecipitated with anti-Cdk2-protein A beads. Cdk2-cyclin activity was assayed as described in “Methods”. The same samples (25 μg) were used for western blot with anti-β-actin antibody to ensure identical loading. Relative intensity (sample vs. LV control) is shown in the lower panel. *, p<0.05. D, Cell lysates described in “C” were separated on 10% SDS gel followed by western blotting with appropriate antibodies as indicated. E. Relative intensity of blots in “D” (sample vs. internal control (β-actin)) was determined by densitometry. *, p<0.05.
Mentions: To dissect the underlying mechanism of MS4a4B-mediated regulation of cell cycle, we analyzed gene expression that regulates cell cycle transition by real-time PCR array. We found that expression of several inhibitory genes was enhanced in MS4a4B-lentivirus-infected EL4 cells, including Ink4 family protein (p16), Cip/Kip family proteins (p21 and p27) and E2f4, the latter having been found to repress gene transcription by interfering with the binding of E2f1, E2f2 and E2f3 to promoters [36]. On the other hand, expression of positive cell cycle regulators, e.g. Cyclin A1, Cyclin B1 and E2f2, was decreased (Fig. 5A and B). Another enhanced inhibitory molecule is Apbb1 (Amyloid beta (A4) precursor protein-binding, family B, member 1), also named Fe65, which is an adaptor protein localized in the nucleus. Overexpression of Fe65 in mouse fibroblasts has been shown to block cell growth by inhibiting the activation of a key S phase gene, the thymidylate synthase (TS) gene [37]. Notably, MS4a4B expression enhanced the production of the calcium/calmodulin-dependent protein kinase (CaM kinase) IIα, also known as Camk2a, whose kinase activity is dependent upon its activation by calmodulin (CaM). CaM is one of the key proteins that transduces a signal in response to increases in intracellular Ca2+. Whether MS4a4B protein is involved in regulation of Ca2+ signaling still remains to be determined. We observed no change in p53 at RNA transcription levels, suggesting that p53 is not involved in MS4a4B-mediated inhibition of cell cycle.

Bottom Line: CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively.In contrast, knockdown of MS4a4B accelerated T cell proliferation.MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America. hui.xu@jefferson.edu

ABSTRACT
MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

Show MeSH
Related in: MedlinePlus