Immune Netw.  2013 Apr;13(2):55-62. 10.4110/in.2013.13.2.55.

Swiprosin-1 Expression Is Up-Regulated through Protein Kinase C-theta and NF-kappaB Pathway in T Cells

• Kim YD
• Kwon MS
• Na BR
• Kim HR
• Lee HS
• Jun CD

• KMID: 1431926
• DOI: http://doi.org/10.4110/in.2013.13.2.55

Abstract

Swiprosin-1 exhibits the highest expression in CD8+ T cells and immature B cells and has been proposed to play a role in lymphocyte biology through actin remodeling. However, regulation of swiprosin-1 gene expression is poorly understood. Here we report that swiprosin-1 is up-regulated in T cells by PKC pathway. Targeted inhibition of the specific protein kinase C (PKC) isotypes by siRNA revealed that PKC-theta is involved in the expression of swiprosin-1 in the human T cells. In contrast, down-regulation of swiprosin-1 by A23187 or ionomycin suggests that calcium-signaling plays a negative role. Interestingly, swiprosin-1 expression is only reduced by treatment with NF-kappaB inhibitors but not by NF-AT inhibitor, suggesting that the NF-kappaB pathway is critical for regulation of swiprosin-1 expression. Collectively, these results suggest that swiprosin-1 is a PKC-theta-inducible gene and that it may modulate the late phase of T cell activation after antigen challenge.

Keyword

T cells; Swiprosin-1; Protein kinase C; Actin remodeling

MeSH Terms

Actins
Biology
Calcimycin
Down-Regulation
Gene Expression
Humans
Ionomycin
Lymphocytes
NF-kappa B
Precursor Cells, B-Lymphoid
Protein Kinase C
Protein Kinases
RNA, Small Interfering
T-Lymphocytes
Actins
Calcimycin
Ionomycin
NF-kappa B
Protein Kinase C
Protein Kinases
RNA, Small Interfering

Figure

• 

  Figure 1 Expression analysis of swiprosin-1 mRNA by RT-PCR. (A) Expression of swiprosin-1 mRNA in Jurkat T cells. Jurkat T cells were stimulated with anti-CD3/CD28, and then the cells were harvested at the indicated time points (0~12 h). The mRNA levels of swiprosin-1 were determined by realtime quantitative PCR (top) and RT-PCR (bottom). GAPDH was used as an internal control. *p<0.01 versus 0 h. (B) Induction of swiprosin-1 in T cells by various stimuli. Jurkat T cells were treated with PMA (200 nM), PHA (1µg/ml), PMA/A23187 (1µM), or PMA/PHA/A23187. At the indicated time points, the expressions of swiprosin-1, IL-2, and IL-3 were determined by RT-PCR. GAPDH was used as an internal control. (C) Western blot analysis of swiprosin-1 expression in T cells. Jurkat T cells were activated by PMA, and then the cells were harvested at the indicated time points (0~24 h). The protein levels were determined by Western blot. All data are from at least three separate experiments. *p<0.01 versus 0 h.

• 

  Figure 2 Swiprosin-1 is up-regulated by PMA but down-regulated by calcium ionophore. (A) Swiprosin-1 is regulated by PKC and calcium modulators. Jurkat T cells were pretreated for 30 min with BAPTAAM (50µM), A23187, or ionomycin (1µM), and then the cells were further incubated with medium alone or PMA, anti-CD3 (10µg/ml), and/or anti-CD28 (2µg/ml). After 6 h of incubation, the mRNA levels were determined as described in Fig. 1A. The protein levels were determined at the 18 h as described in Fig. 1C. Data represent three separate experiments. The data shown in the bar graphs represent mean±SD values of triplicate experiments. *p<0.01 versus PMA alone. (B) Jurkat T cells were pretreated for 30 min with MAP kinase inhibitors including SB203580 (10µM), PD098059 (10µM), and SP600125 (10µM), and then the cells were further stimulated for 6 h with PMA. The mRNA levels were determined as described in Fig. 1A. (C) Jurkat T cells were pretreated for 30 min with PKC inhibitors including Rottlerin (10µM), staurosporine (STSN, 500 nM), Gö6976 (100 nM), and Gö6983 (100 nM), and then the cells were further stimulated for 6 h with PMA. The mRNA levels were determined as described in Fig. 1A. Data represent three separate experiments. *p<0.01 versus PMA alone.

• 

  Figure 3 Swiprosin-1 expression is dependent on PKC-θ pathways in T cells. (A) Detection of PKC isoforms by Western blot in Jurkat T cells. (B) Knock-down of PKC isoforms by specific siRNA. Jurkat T cells were transfected with siRNA targeting specific PKC isoforms. At the indicated time points, the expression of PKC isoforms was analyzed by Western blot. Blots were also probed with antibody to β-actin to confirm an equal loading. (C) Knock-down of PKC-θ inhibits phorbol ester-induced swiprosin-1 expression in Jurkat T cells. After 72 h of siRNA transfection against indicated PKC isoforms, cells were treated with or without PMA (200 nM) for 6 h and the expression of swiprosin-1 was analyzed by RT-PCR (top) and quantitative RT-PCR (bottom). Amplification of GAPDH was used as an internal control. Data represent three separate experiments. The data shown in the bar graphs represent mean±SD values of triplicate experiments. *p<0.01 versus scrambled siRNA. SC, scrambled.

• 

  Figure 4 Swiprosin-1 expression is regulated by the PKC-θ pathway in Jurkat T and human primary PBLs. (A and B) Jurkat T cells (A) and primary PBLs (B) were stimulated with anti-CD3/CD28, and then the cells were harvested and the mRNA levels were determined as described by Fig. 1A. The expression of PKC isoforms in primary T cells was analyzed by Western blot. Data represent three separate experiments. The data shown in the bar graphs represent mean±SD values of triplicate experiments. *p<0.01 versus scrambled siRNA. SC, scrambled.

• 

  Figure 5 Swiprosin-1 expression is regulated by the NF-κB but not by the NF-AT. (A) Jurkat T cells were transiently transfected with NF-κB or NF-AT luciferase reporter constructs. After 24 h, the cells were treated with PMA, A23187, or PMA/A23187 and incubated for 12 h. NF-κB- or NF-AT-dependent transcriptional activities were determined by luciferase activity assay. Data are representative of three separate experiments. The data shown in bar graphs represent mean±SD values of triplicate experiments. (B) Jurkat T cells were pretreated for 30 min with NF-κB inhibitors including MG132 (1µM), and CAPE (10µM) or NF-AT inhibitor CsA (1µg/ml), and then the cells were further stimulated for 6 h with PMA (left) or PMA/A23187 (right). The mRNA levels were determined as described in Fig. 1A. Data represent three separate experiments.

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