Immune Netw.  2015 Oct;15(5):260-267. 10.4110/in.2015.15.5.260.

Engagement of CD99 Reduces AP-1 Activity by Inducing BATF in the Human Multiple Myeloma Cell Line RPMI8226

• Gil M
• Pak HK
• Park SJ
• Lee AN
• Park YS
• Lee H
• Lee H
• Kim KE
• Lee KJ
• Yoon DH
• Chung YS
• Park CS

• KMID: 2150855
• DOI: http://doi.org/10.4110/in.2015.15.5.260

Abstract

CD99 signaling is crucial to a diverse range of biological functions including survival and proliferation. CD99 engagement is reported to augment activator protein-1 (AP-1) activity through mitogen-activated protein (MAP) kinase pathways in a T-lymphoblastic lymphoma cell line Jurkat and in breast cancer cell lines. In this study, we report that CD99 differentially regulated AP-1 activity in the human myeloma cell line RPMI8226. CD99 was highly expressed and the CD99 engagement led to activation of the MAP kinases, but suppressed AP-1 activity by inducing the expression of basic leucine zipper transcription factor, ATF-like (BATF), a negative regulator of AP-1 in RPMI8226 cells. By contrast, engagement of CD99 enhanced AP-1 activity and did not change the BATF expression in Jurkat cells. CD99 engagement reduced the proliferation of RPMI8226 cells and expression of cyclin 1 and 3. Overall, these results suggest novel CD99 functions in RPMI8226 cells.

Keyword

CD99; BATF; AP-1; Proliferation; MAP kinase

MeSH Terms

Breast Neoplasms
Cell Line*
Cyclins
Humans*
Jurkat Cells
Leucine Zippers
Lymphoma
Multiple Myeloma*
Phosphotransferases
Transcription Factor AP-1*
Transcription Factors
Cyclins
Phosphotransferases
Transcription Factor AP-1
Transcription Factors

Figure

• 

  Figure 1 Expression of CD99 in B-cell lines. (A) Surface expression of CD99 in B-cell lines. Cells were stained using an anti-CD99 Ab (DN16-PE, solid line) or isotype control Ab (dotted line) and measured using flow cytometry. (B) PCR analysis with primers designed to specifically amplify CD99 type-I and -II. As described previously, the 583 bp fragment is characteristic of CD99 type-I and the 515 bp fragment is characteristic of CD99 type-II (513). Levels of GAPDH mRNA were used to normalize expression.

• 

  Figure 2 CD99 engagement reduces AP-1 activity and induces BATF expression in RPMI8226 cells. (A) AP-1 activity in RPMI8226 and Jurkat cells upon CD99 engagement. Both AP-1 luciferase constructs and a β-galactosidase expression plasmid were transfected into RPMI8226 and Jurkat cells. Luciferase activity was measured 24 hours after CD99 engagement and normalized against β-galactosidase activity. (B) Activation of ERK and JNK upon CD99 engagement in RPMI8226 cells. RPMI8226 cells were stimulated with 10 µg/ml of an anti-CD99 Ab (YG32) at 37℃ for 15, 30, and 60 min. The activities of ERK, JNK, and p38 kinase were measured by western blotting with antibodies specific for the phosphorylated form of each kinase, as well as suitable controls. Results are from a representative experiment performed in triplicate. (C) Expression of AP-1 transcription factors was measured using real-time PCR at 72 hours after CD99 engagement. Expression levels of the transcript in CD99-engaged cells are shown relative to those in control Ab (mouse IgG)-treated cells. (D) BATF expression was measured using real-time PCR at 1, 3, 8, and 24 hours after CD99 engagement in RPMI8226 cells. (E) BATF expression in CD99-engaged and control Jurkat cells. BATF expression was measured 24 hours after CD99 engagement by real-time PCR.

• 

  Figure 3 BATF-mediated regulation of AP-1 activity in RPMI8226 cells. (A) Overexpression of BATF reduces AP-1 activity in RPMI8226 and 293T cells. Cells were transiently transfected with reporter plasmids and expression plasmids, and treated with YG32. AP-1 activity was measured using a luciferase assay. Luciferase activity in cells co-transfected with pcDNA3.1-BATF is shown relative to that in cells co-transfected with pCDNA3.1 (set to 1). (B) Expression of BATF in RPMI8226 cells was reduced by siRNA treatment. Expression of BATF was measured using real-time PCR at 2 days after electroporation of siRNA oligonucleotides. (C) BATF knockdown reverses the reduction in AP-1 activity upon CD99 engagement. Cells were transfected with reporter plasmids 2 days after electroporation with siRNA oligonucleotides. Luciferase activities were normalized with respect to -galactosidase activities. The values show this normalized activity in Ab-treated cells relative to that in cells treated with control IgG. The error bars indicate the SD of three independent experiments.

• 

  Figure 4 CD99 engagement reduces the proliferation of RPMI8226 cells. (A) The number of RPMI8226 cells was counted using a hemocytometer after 3 days of treatment with an anti-CD99 Ab (YG32) or control IgG. The asterisk denotes a significant difference (p<0.05) between the two groups. (B) CFSE-labeled RPMI8226 cells at day 0 (filled histogram) were incubated for 3 days with control IgG (dotted line) or YG32 (solid line), and analyzed by flow cytometry. (C) RPMI8226 cells were cultured with YG32 or control IgG for 24 hours, stained with TMRE, and quantified using flow cytometry. (D) Annexin V-positive cells were quantified using flow cytometry after culture for the indicated amount of time in the presence of control IgG, YG32, or bortezomib. The ratio of the number of annexin V-positive cells to the total number of cells is shown (E) Expression of cyclin D1, D2, and D3 was measured by performing real-time PCR using the cDNA of RPMI8226 cells harvested after 3 days of incubation with YG32.

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