Hippocalcin Promotes Neuronal Differentiation and Inhibits Astrocytic Differentiation in Neural Stem Cells

Abstract

Hippocalcin (HPCA) is a calcium-binding protein that is restricted to nervous tissue and contributes to neuronal activity. Here we report that, in addition to inducing neurogenesis, HPCA inhibits astrocytic differentiation of neural stem cells. It promotes neurogenesis by regulating protein kinase Cα (PKCα) activation by translocating to the membrane and binding to phosphoinositide-dependent protein kinase 1 (PDK1), which induces PKCα phosphorylation. We also found that phospholipase D1 (PLD1) is implicated in the HPCA-mediated neurogenesis pathway; this enzyme promotes dephosphorylation of signal transducer and activator of transcription 3 (STAT3[Y705]), which is necessary for astrocytic differentiation. Moreover, we found that the SH2-domain-containing tyrosine phosphatase 1 (SHP-1) acts upstream of STAT3. Importantly, this SHP-1-dependent STAT3-inhibitory mechanism is closely involved in neurogenesis and suppression of gliogenesis by HPCA. Taken together, these observations suggest that HPCA promotes neuronal differentiation through activation of the PKCα/PLD1 cascade followed by activation of SHP-1, which dephosphorylates STAT3(Y705), leading to inhibition of astrocytic differentiation.

Keywords: SH2-domain-containing tyrosine phosphatase-1 (SHP-1); gliogenesis; hippocalcin (HPCA); neural stem cells; neurogenesis; phosphoinositide-dependent protein kinase-1 (PDK1); phospholipase D1 (PLD1); signal transducers and activator of transcription 3 (STAT3).

Figure 1

Effect of HPCA Expression during Neuronal Differentiation of NSCs (A) HPCA immunostaining of coronal sections of E14 rat brain cortex. The boxed area is magnified. Scale bar, 20 μm. (B) Neuronal differentiation was induced by removal of bFGF for 1 day, and Hpca mRNA expression was analyzed by RT-PCR. Twenty micrograms of protein was analyzed by western blotting with anti-HPCA and anti-β-ACTIN. (C and D) NSCs were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and allowed to differentiate for 24 hr. mRNA levels of neuronal factors were analyzed by RT-PCR (C) and real-time RT-PCR (D). ^∗p < 0.05 compared with the −bFGF/vector control (mean ± SD; n = 3). (E) Cells were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and induced to differentiate for 24 hr. Levels of TUJ1, GFAP, MYC, and β-ACTIN were determined by western blot. ^∗p < 0.05 compared with the −bFGF/vector control (mean ± SD; n = 3). (F) Cells were transduced with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc and induced to differentiate by withdrawal of bFGF. After 3 days, GFP-positive cells were examined by fluorescence microscopy and stained with anti-EGFP (green) and anti-TUJ1 (red). Scale bar, 20 μm. (G and H) Neurite lengths were measured and the proportions of TUJ1-positive cells and total cells were determined in randomly selected areas from at least three slides of each condition. ^∗p < 0.05 compared with the −bFGF/vector control (mean ± SD; n = 3).

Figure 2

Increased Intracellular Ca²⁺ Affects HPCA-PKCα Activation during Neuronal Differentiation of NSCs (A and B) NSCs were used for fluo-3/AM Ca²⁺ imaging 1 day after plating on poly-L-lysine-coated glass coverslips (see Experimental Procedures). We collected images every 1 s at 470 nm excitation/514 nm emission using Metafluor software. The threshold of increased intracellular Ca²⁺ was defined as a 20% increase from the basal level. (C) Cells were depleted with bFGF for 5 min, fixed, and immunostained with anti-HPCA (red). The boxed area is magnified. Scale bar, 5 μm. (D and E) Cells were pretreated with 500 μM EGTA for 1 day and incubated for 3 days after removal of bFGF. After immunostaining with anti-TUJ1, TUJ1-positive cells were counted under fluorescence microscopy. The proportion of TUJ1-positive cells and total cells was determined in random areas from at least three slides of each condition. Data are shown as mean ± SD (n = 3). N.S, not significant (p = 0.245). Scale bar, 20 μm. (F) Schematic of Hpca (wild-type) and its deletion mutants. (G and H) NSCs were transfected with pMSCV-IRES-EGFP, pMSCV-IRES-EGFP-Hpca-Myc, pMSCV-IRES-EGFP-Hip_1-72-Myc, or pMSCV-IRES-EGFP-Hip_65-193-Myc for 48 hr and induced to differentiate for 24 hr. mRNA levels of neuronal factors were analyzed by RT-PCR (G) and real-time RT-PCR (H). ^∗p < 0.05 compared with −bFGF/Hpca (mean ± SD; n = 3). (I and J) G2 is a key residue for myristoylation of HPCA. NSCs were transfected with pMSCV-IRES-EGFP, pMSCV-IRES-EGFP-Hpca-Myc, or pMSCV-IRES-EGFP-Hpca G2A-Myc for 48 hr and induced to differentiate for 24 hr. mRNA levels of neuronal factors were analyzed by RT-PCR (I) and real-time RT-PCR (J). ^∗p < 0.05 compared with −bFGF/Hpca (mean ± SD; n = 3). (K) Cells were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and induced to differentiate by withdrawal of bFGF for 24 hr. Cells were then lysed and centrifuged at 100,000 × g to separate cytosolic and membrane fractions. Samples of protein (30 μg) from each fraction were analyzed by western blotting with anti-PKCα, anti-MYC, anti-integrin α2, and anti-GAPDH. Data are shown as mean ± SD (n = 3). (L) Cells were transfected with p-MSCV-IRES-EGFP-Hpca-Myc for 48 hr, and induced differentiate for 24 hr. Immunoprecipitates (IP) from the cell lysates with anti-MYC were analyzed by western blotting with anti-PKCα and anti-MYC. (M) Cells were transiently transfected with control siRNA or Hpca siRNA for 48 hr, then incubated for 1 day after removal of bFGF. Proteins were analyzed by western blotting with anti-p-PKCα, anti-PKCα, anti-HPCA, and anti-GAPDH. ^∗p < 0.05 compared with the −bFGF/control siRNA (mean ± SD; n = 3). (N) Cells were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and then treated with 10 μM RO320432 for 30 min before differentiation for 1 day. Cells were lysed and analyzed by western blotting with anti-TUJ1, anti-GFAP, anti-MYC, and anti-GAPDH. ^∗p < 0.05 compared with −bFGF/Hpca (mean ± SD; n = 3).

Figure 3

PDK1 Is Involved in HPCA-Mediated PKCα Activation and Neuronal Differentiation of NSCs (A) NSCs were pretreated with 5 μM GSK2334470 or 10 μM BAG956 for 30 min before differentiation for 1 day. Cells were lysed and analyzed by western blotting with anti-p-PKCα, anti-PKCα, anti-p-PDK1, anti-PDK1, and anti-GAPDH. (B and C) Cells were pretreated with 5 μM GSK 2334470 and allowed to differentiate for 1 day. mRNA levels of neuronal factors were analyzed by RT-PCR (B) and real-time RT-PCR (C). ^∗p < 0.05 compared with the −bFGF control (mean ± SD; n = 3). (D) Cells were pretreated with 5 μM GSK2334470 and induced to differentiate by withdrawal of bFGF. After differentiation for 3 days, fixed cells were immunostained with anti-TUJ1 (green) and anti-GFAP (red). Scale bar, 20 μm. (E and F) Neurite lengths were measured and the numbers of TUJ1-positive cells and total cells were determined in randomly selected areas from at least three slides of each condition. ^∗p < 0.05 compared with the −bFGF control (mean ± SD; n = 3). (G) Cells were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr, and induced to differentiate for 24 hr. Immunoprecipitates from the cell lysates with anti-PDK1 were analyzed by western blotting with anti-p-PKCα, anti-PKCα, anti-PDK1, and anti-MYC.

Figure 4

PLD1 Is Required for Hpca-Induced Neuronal Differentiation of NSCs (A) Coronal sections of E14 rat brain cortex immunostained with anti-HPCA and anti-PLD1. Scale bar, 10 μm. (B) NSCs were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and labeled with 2 μCi/mL [³H]palmitic acid. They were pretreated with 10 μM RO320432 for 30 min and allowed to differentiate for 1 day. PLD activities were determined by measuring the formation of [³H]PBt in the presence of 1-butanol. ^∗p < 0.05 compared with −bFGF/Hpca (mean ± SD; n = 5). (C) Cells were transfected with control siRNA or Hpca siRNA for 48 hr and then incubated for 1 day after removal of bFGF. Proteins were analyzed by western blotting with anti-p-PLD1 (T147), anti-PLD1, anti-HPCA, and anti-β-ACTIN. (D) Cells were transfected with control siRNA, Hpca siRNA, or Pld1 siRNA for 48 hr and induced to differentiate through withdrawal of bFGF. After differentiation for 3 days, cells were stained for immunocytochemical analysis of neuronal (TUJ1, green) and astrocytic (GFAP, red) markers. Scale bar, 10 μm. Graphs show the percentages of TUJ1- and GFAP-positive cells. Data are shown as mean ± SD (n = 3). ^∗Significantly different from −bFGF/control siRNA at p < 0.05 (for TUJ1). ^†Significantly different from −bFGF/control siRNA at p < 0.05 (for GFAP). (E) Cells were transfected with control siRNA, Hpca siRNA, or Pld1 siRNA for 48 hr and induced to differentiate by withdrawal of bFGF. After differentiation for 3 days, fixed cells were immunostained with anti-TUJ1. Scale bar, 10 μm. (F) The numbers of spines per 10-μm dendrite were counted in randomly selected areas from at least three slides of each condition. Data are shown as mean ± SD (n = 5). ^∗p < 0.05 compared with the −bFGF/control siRNA. ^∗∗p < 0.01 compared with the −bFGF/control siRNA.

Figure 5

Functional Analysis of STAT3(Y705) Activity in HPCA-Mediated Neuronal Differentiation (A) NSCs were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and induced to differentiate for 1 day. Cell lysates were analyzed by western blotting with anti-p-STAT3(Y705), anti-p-STAT3 (S727), anti-STAT3, anti-MYC, and anti-GAPDH. Data are shown as mean ± SD (n = 3). ^∗p < 0.05 compared with the +bFGF/vector control. ^∗∗p < 0.05 compared with the −bFGF/vector control. (B) Cells were transfected with control siRNA or Hpca siRNA for 48 hr and allowed to differentiate for 1 day. Proteins were analyzed by western blotting with anti-p-STAT3(Y705), anti-STAT3, anti-TUJ1, anti-GFAP, and anti-GAPDH. ^∗p < 0.05 compared with the −bFGF/control siRNA (mean ± SD; n = 3). (C) Diagrams of Stat3WT (wild-type) and Stat3YF (mutant). CC, coiled-coil domain; DBD, DNA-binding domain; LK, linker domain; SH2, Src homology 2; TA, transcriptional activation domain. (D) Vector (pBOS-FLAG), Stat3WT, or Stat3YF were transfected into NSCs for 48 hr, and the cells were induced to differentiate for 2 days. Cells were lysed and analyzed by western blotting with anti-p-STAT3(Y705), anti-STAT3, anti-TUJ1, anti-GFAP, and anti-GAPDH. (E and F) Cells were transfected with Stat3WT or Stat3YF for 48 hr. After differentiation for 3 days, cells were stained for neuronal (TUJ1, green) and astrocytic (GFAP, red) markers. Scale bar, 10 μm. Graphs show the percentages of TUJ1- and GFAP-positive cells. Data are shown as mean ± SD (n = 3). ^∗Significantly different from −bFGF/Stat3WT at p < 0.05 (for TUJ1). ^†Significantly different from −bFGF/Stat3WT at p < 0.05 (for GFAP). (G) Neurite lengths were measured in randomly selected areas from at least three slides of each condition. ^∗p < 0.05 compared with −bFGF/Stat3WT (mean ± SD; n = 3). (H) Cells infected with retroviruses expressing pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc were transiently transfected with Stat3WT or Stat3YF for 48 hr. The cells were induced to differentiate for 2 days. Levels of TUJ1 and GFAP were determined by western blotting. Data are shown as mean ± SD (n = 3). ^∗p < 0.05, ^∗∗p < 0.01. (I and J) Cells were treated as in (H) and induced to differentiate for 3 days. Cells were stained for immunocytochemical analysis of neuronal (TUJ1, green) and astrocytic (GFAP, red) markers. Scale bar, 10 μm. Graphs show the percentages of TUJ1- and GFAP-positive cells. Data are shown as mean ± SD (n = 3). ^∗Significantly different from −bFGF/Stat3WT/Hpca at p < 0.05 (for TUJ1). ^†Significantly different from −bFGF/Stat3WT/Hpca at p < 0.05 (for GFAP). (K) Neurite lengths were measured in randomly selected areas from at least three slides of each condition. ^∗p < 0.05 compared with −bFGF/Stat3WT/Hpca (mean ± SD; n = 3).

Figure 6

Dephosphorylation of STAT3(Y705) by SHP-1 Is Related to the Neuronal Differentiation of NSCs (A) NSCs were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr before pretreatment with 1 μM PTPI(1) for 30 min. Cells were then allowed to differentiate for 1 day before lysis and analysis by western blotting with anti-p-STAT3(Y705), anti-STAT3, anti-TUJ1, anti-GFAP, anti-MYC, and anti-GAPDH. ^∗p < 0.05 compared with −bFGF/Hpca (mean ± SD; n = 3). (B) Cells were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and then treated with 1 μM PTPI(1) for 30 min before differentiation for 1 day. Fixed cells were immunostained with anti-STAT3 (red) and anti-DAPI (blue); STAT3 nuclei were counted. Arrows indicate magnified cells. Graphs show the ratio of STAT3 nuclei to total nuclei in random areas from at least three slides of each condition. ^∗p < 0.05 compared with −bFGF/Hpca (mean ± SD; n = 3). (C and D) Cells were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and pretreated with 1 μM PTPI(1) for 30 min. After differentiation for 3 days, cells were analyzed immunocytochemically using neuronal (TUJ1, green) and astrocytic (GFAP, red) markers. Scale bar, 50 μm. Graphs show the percentages of TUJ1- and GFAP-positive cells. Data are shown as mean ± SD (n = 3). ^∗Significantly different from −bFGF/Hpca at p < 0.05 (for TUJ1). ^†Significantly different from −bFGF/Hpca at p < 0.05 (for GFAP). (E) Cells were transfected with control siRNA or Shp-1 siRNA for 48 hr and allowed to differentiate for 1 day. Proteins were analyzed by western blotting with anti-p-STAT3(Y705), anti-STAT3, anti-SHP-1, and anti-GAPDH. ^∗p < 0.05 compared with the −bFGF/control siRNA (mean ± SD; n = 3). (F) Cells were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and induced to differentiate for 24 hr. Anti-SHP-1 immunoprecipitates from the cell lysates were analyzed by western blotting with anti-STAT3, anti-p-STAT3, anti-SHP-1, and anti-MYC.

Figure 7

SHP-1 Is Required for Neuronal Differentiation of NSCs (A) NSCs were treated with 10 μM or 20 μM phosphatidic acid (PA) for 1 day in the presence of bFGF. Cell lysates were analyzed by western blotting with anti-p-SHP-1, anti-SHP-1, anti-p-STAT3(Y705), anti-STAT3, and anti-GAPDH. (B) Cells were transfected with control siRNA or Pld1 siRNA for 48 hr and allowed to differentiate for 1 day. Proteins were analyzed by western blotting with anti-p-SHP1 (Y536), anti-SHP-1, anti-PLD1, and anti-GAPDH. ^∗p < 0.05 compared with the −bFGF/control siRNA (mean ± SD; n = 3). (C) Cells were transfected with control siRNA or Hpca siRNA for 48 hr and allowed to differentiate for 1 day. Cell lysates were analyzed by western blotting with anti-p-SHP1 (Y536), anti-SHP-1, anti-HPCA, and anti-GAPDH. ^∗p < 0.05 compared with the −bFGF/control siRNA (mean ± SD; n = 3). (D) Cells were transfected with control siRNA, Hpca siRNA, or Pld1 siRNA for 48 hr and allowed to differentiate for 1 day. SHP-1 activity was measured by phosphatase activity assay (see Experimental Procedures). ^∗p < 0.05 compared with the −bFGF/control siRNA (mean ± SD; n = 5). (E and F) Cells were transfected with control siRNA or Shp-1 siRNA for 48 hr and allowed to differentiate for 3 days. Fixed cells were immunostained with anti-TUJ1 and anti-GFAP, and TUJ1- and GFAP-positive cells were counted. Scale bar, 20 μm. Graphs show the percentages of TUJ1- and GFAP-positive cells. Data are shown as mean ± SD (n = 5). ^∗Significantly different from −bFGF/control siRNA at p < 0.05 (for TUJ1). ^†Significantly different from −bFGF/control siRNA at p < 0.05 (for GFAP). (G) Neurite lengths were measured in randomly selected areas from at least three slides of each condition. ^∗p < 0.05 compared with the −bFGF/control siRNA (mean ± SD; n = 3). (H) Cells were transfected with control siRNA or Shp-1 siRNA for 48 hr and induced to differentiate by withdrawal of bFGF. After differentiation for 3 days, fixed cells were immunostained with anti-TUJ1. Scale bar, 10 μm. The numbers of spines per 10-μm dendrite were counted in randomly selected areas from at least three slides of each condition. ^∗p < 0.05 compared with the −bFGF/control siRNA (mean ± SD; n = 3). (I) The model suggests that HPCA plays a crucial role in neurogenesis through modulation of the PKCα/PLD1/SHP-1/STAT3 signaling pathway.