Regulation of cell surface protease receptor S100A10 by retinoic acid therapy in acute promyelocytic leukemia (APL)☆

Abstract

S100A10 (p11), a member of the S100 family of small dimeric EF-hand-type Ca²⁺-binding proteins, plays a role in a variety of both intracellular and extracellular processes. Previous studies have suggested that p11 is intrinsically unstable and requires binding to annexin A2 (p36) to prevent its rapid ubiquitylation and degradation. Our laboratory has shown that p11 levels are stimulated by the expression of the oncoprotein, PML/RARα. Furthermore, treatment of the APL cell line, NB4 with all-trans retinoic acid (ATRA) causes the rapid loss of p36 and p11 protein. However, the mechanism by which ATRA regulates p11 levels has not been established. Here, we show that the proteasomal inhibitor, lactacystin reversed the ATRA-dependent loss of p11, but did not cause an accumulation of ubiquitylated forms of p11, suggesting that ATRA promotes the proteasomal degradation of p11 in an ubiquitin-independent manner. ATRA treatment of MCF-7 breast cancer cells reduced p11 but not p36 transcript and protein levels, thus indicating that ATRA can regulate p11 levels independently of PML/RARα and p36. Overexpression of p36 upregulated p11 protein but not mRNA levels, indicating that p36 affects p11 post translationally. The forced expression of ubiquitin and p11 in 293 T cells resulted in ubiquitylation of p11 that was blocked by mutagenesis of lysine 57. This study highlights the complex regulation of p11 by retinoid signaling and challenges the hypothesis that ubiquitin-mediated proteasomal degradation of p11 represents a universal mechanism of regulation of this protein.

Fig. 1. ATRA induces the ubiquitin-independent proteasomal degradation of p11 in APL cell line, NB4.

a Immunoblot analysis of NB4 cells treated for 48 h with 1 µM ATRA alone or in combination with 2 µM lactacystin (LC) or 2 µM PYR-41. b Immunoprecipitation of p11 or IgG1 isotype control in NB4 cells treated for 24 h with 1 µM ATRA alone or in combination with 2 µM LC. Purified AIIt (0.25 µg) was used as a control. c Immunoprecipitation of p11 in PR9 cells treated for 48 h with 2 µM LC. Purified AIIt (0.25 µg) was used as a control. d Immunoprecipitation of p11 or IRS-1 in NB4 cells treated for 24 h with 1 µM ATRA alone, or in combination with 2 µM LC. Cell lysates were prepared and the level of the indicated proteins were examined by immunoblot analysis with β-actin used as a loading control. Data is expressed as the mean ± S.D. of three independent experiments. Statistical significance was determined using one-way ANOVA (with Tukey multiple comparisons), where **P < 0.01, ***P < 0.001, and ****P < 0.0001 are considered statistically significant

Fig. 2. Lysine 57 is the site of ubiquitylated of p11, but may not be involved in proteasomal degradation.

a Immunoblot analysis of HEK293T cells transiently transfected using pcDNA3.1-empty vector or pcDNA3.1-p11 vector alone or treated for 18 h using 3 µM lactacystin (LC), 1 mM NH₄Cl, 1 µM MDL28170, or 1 µM calpain inhibitor IV (C.I.4). b Immunoblot analysis of HEK293T cells transiently transfected using pcDNA3.1-empty vector or pcDNA3.1-p11 vector alone or in combination with pRK5-HA-ubiquitin wild-type (ub-WT) or mutant, lysine-less ubiquitin (ub-K0). c Immunoblot analysis of HEK293T cells transiently expressing p11-wild type (p11WT) or p11 mutants (with Lys54 or Lys57 changed to arginine: p11K54R and p11K57R, respectively) alone or in combination with pRK5-HA-ub-K0. Cell lysates were prepared and the levels of the indicated proteins were examined by immunoblot analysis with β-actin used as a loading control. Data is expressed as the mean ± S.D. of three independent experiments. Statistical significance was determined using one-way ANOVA (with Tukey multiple comparisons), where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 are considered statistically significant

Fig. 3. ATRA downregulates p11 and p36 expression through the loss of PML-RARα expression in APL cells.

Cell lysates were prepared from (a) NB4 or NB4-MR2 were treated without or with 1 µM ATRA for 72 h (b) ATRA-treated (1 µM for 48 h) HL-60 and U937 cells, (c) PR9 cell treated with or without 100 µM zinc sulfate (ZnSO₄) for 48 h, (d) ATRA-treated (1 µM for 48 h) PR9 ( ± 100 µM ZnSO₄) for 48 h, and (e) MCF-7 cells treated for 48 h with 1 µM ATRA alone or in combination with 2 µM lactacystin (LC) or 2 µM PYR-41. Cell lysates were prepared and the levels of the indicated proteins were examined by immunoblot analysis with β-actin used as a loading control. The data are expressed as the mean ± S.D. of three independent experiments. Statistical significance was determined using one-way ANOVA (with Tukey multiple comparisons), where **P < 0.01, ***P < 0.001 and ****P < 0.0001 are considered statistically significant

Fig. 4. Transcript levels of p11 and p36 are regulated by PML-RARα and ATRA treatment.

Total RNA extracted from (a) PR9 cells treated (48 h) without or with 100 µM ZnSO₄, (b) NB4 cells treated (48 h) without or with 1 µM ATRA alone or in combination with 2 µM LC, and (c) ATRA-treated (48 h) MCF-7 cells. The relative expression of p11 and p36 mRNA levels was determined from cDNA (25 ng) by qPCR analysis and normalized to GAPDH, β-actin and HPRT1. Data is expressed as the mean ± S.D. of three (b) or four (a, c) independent experiments. Statistical significance was determined using (a, c) the Student t test for unpaired observations or (b) one-way ANOVA (with Tukey multiple comparisons), where *P < 0.05, **P < 0.01, and ****P < 0.0001 are considered statistically significance

Fig. 5. P36 upregulates p11 protein without affecting transcript levels.

Immunoblot analysis of (a) peritoneal macrophages isolated from p36^+/+ and p36^−/− mice treated for 24 h using 3 μM LC or 10 μM PYR-41 and (b) HEK293T cells transiently transfected using pcDNA3.1-empty vector or pcDNA3.1-p36 vector. Cell lysates were prepared and the levels of the indicated proteins were examined by immunoblot analysis with β-actin used as a loading control. Total RNA extracted from (c) HEK293T cells transiently transfected using pcDNA3.1-empty vector or pcDNA3.1-p36 vector were used for cDNA synthesis. The relative expression of p11 and p36 mRNA levels was determined from cDNA (25 ng) by qPCR analysis and normalized to GAPDH, β-actin and HPRT1. d Immunoblot analysis of HEK293T cells treated for 16 h using 2.5 µM lactacystin (LC). Cell lysates were prepared and the levels of the indicated proteins were examined by immunoblot analysis with β-actin used as a loading control. The data are expressed as the mean ± S.D. of three independent experiments. Statistical significance was determined using (b, c) the Student t test for unpaired observations or (a, d) one-way ANOVA (with Tukey multiple comparisons), where **P < 0.01 and ****P < 0.0001 are considered statistically significant