SH2B1beta (SH2-Bbeta) enhances expression of a subset of nerve growth factor-regulated genes important for neuronal differentiation including genes encoding urokinase plasminogen activator receptor and matrix metalloproteinase 3/10

Abstract

Previous work showed that the adapter protein SH2B adapter protein 1beta (SH2B1) (SH2-B) binds to the activated form of the nerve growth factor (NGF) receptor TrkA and is critical for both NGF-dependent neurite outgrowth and maintenance. To identify SH2B1beta-regulated genes critical for neurite outgrowth, we performed microarray analysis of control PC12 cells and PC12 cells stably overexpressing SH2B1beta (PC12-SH2B1beta) or the dominant-negative SH2B1beta(R555E) [PC12-SH2B1beta(R555E)]. NGF-induced microarray expression of Plaur and Mmp10 genes was greatly enhanced in PC12-SH2B1beta cells, whereas NGF-induced Plaur and Mmp3 expression was substantially depressed in PC12-SH2B1beta(R555E) cells. Plaur, Mmp3, and Mmp10 are among the 12 genes most highly up-regulated after 6 h of NGF. Their protein products [urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase 3 (MMP3), and MMP10] lie in the same pathway of extracellular matrix degradation; uPAR has been shown previously to be critical for NGF-induced neurite outgrowth. Quantitative real-time PCR analysis revealed SH2B1beta enhancement of NGF induction of all three genes and the suppression of NGF induction of all three when endogenous SH2B1 was reduced using short hairpin RNA against SH2B1 and in PC12-SH2B1beta(R555E) cells. NGF-induced levels of uPAR and MMP3/10 and neurite outgrowth through Matrigel (MMP3-dependent) were also increased in PC12-SH2B1beta cells. These results suggest that SH2B1beta stimulates NGF-induced neuronal differentiation at least in part by enhancing expression of a specific subset of NGF-sensitive genes, including Plaur, Mmp3, and/or Mmp10, required for neurite outgrowth.

Figure 1

SH2B1β Enhances a Subset of NGF-Responsive Genes A, The expression of 511 genes and ESTs (588 probe sets) was at least doubled by treatment of control PC12 cells with NGF (6 h, 100 ng/ml) (C+/C− ≥ 2). Among the 511 genes and ESTs, the NGF-induced expression of 34 genes and ESTs was enhanced by at least 100% by overexpression of SH2B1β, represented by the white circle (C+/C− ≥ 2 and ΔS/ΔC ≥ 2 where ΔS = S+/S− and ΔC = C+/C−). Among the 511 genes and ESTs, the NGF-induced expression of 153 genes was reduced by 50% or more in cells overexpressing SH2B1β(R555E), represented by the dark gray circle (C+/C− ≥ 2 and ΔR/ΔC ≤ 0.5 where ΔR = R+/R− and ΔC = C+/C−). There were only two genes and four ESTs that matched both criteria, represented by the overlapping area between the white and dark gray circles (C+/C− ≥ 2; ΔS/ΔC ≥ 2; ΔR/ΔC ≤ 0.5). B, The expression of 513 genes and ESTs (507 probe sets) was reduced by at least 50% by treatment with NGF (6 h, 100 ng/ml) (C+/C− ≤ 0.5). Among the 513 genes and ESTs, the expression of 49 genes and ESTs was further reduced by overexpression of SH2B1β, represented by the white circle (C+/C− ≤ 0.5 and ΔS/ΔC ≤ 0.5). The NGF-induced inhibition of 105 genes and ESTs was, in addition, dampened by overexpression of SH2B1β(R555E), represented by the dark gray circle (C+/C− ≤ 0.5 and ΔR/ΔC ≥ 2). There was only one EST that matched both criteria, represented by the overlapping area between the white and dark gray circles (C+/C− ≤ 0.5; ΔS/ΔC ≤ 0.5; ΔR/ΔC ≥ 2). C−, S−, and R− represent gene expression levels in control PC12, PC12-SH2B1β, and PC12-SH2B1β(R555E) cells, respectively, in the absence of NGF. C+, S+ and R+ represent gene expression levels in NGF-treated control PC12, PC12-SH2B1β, and PC12-SH2B1β(R555E) cells, respectively. ΔC = C+/C−; ΔS = S+/S−; ΔR = R+/R−.

Figure 2

Functional Classification of NGF-Responsive Genes in Control PC12, PC12-SH2B1β, and PC12-SH2B1β(R555E) Cells Genes that were either up-regulated to at least twice the basal value by NGF (A) or down-regulated more than 50% by NGF (B) in control PC12, PC12-SH2B1β, and PC12-SH2B1β(R555E) cells were grouped into gene ontology categories. Overrepresented categories with P < 0.05 are shown.

Figure 3

NGF-Responsive Genes that Are Highly Regulated by SH2B1β A, NGF-induced gene expression profiles in control PC12 (C), PC12-SH2B1β (SH2B1β), and PC12-SH2B1β(R555E) (R555E) cells were clustered and visualized using Eisen software (http://rana.lbl.gov/EisenSoftware.htm). Four different clusters of genes that show NGF-induced gene expression in all three cell types are shown. For each gene, black represents the average value of all six probe sets. The relative intensity of red represents the degree of increased mRNA levels, and the relative intensity of green represents the degree of decreased mRNA levels. The number of arrows represents the relative levels of increase. B, Microarray data for selected genes from each of the four clusters. C, Gene expression levels for six of the genes shown in A were verified by QT-PCR, normalized first to levels of GAPDH gene expression and then to levels of gene expression seen in control PC12 cells treated with NGF. Means ± sem from three to four experiments are shown. *, Statistically significant differences (P < 0.05) using a one-tailed, paired Student’s t test.

Figure 4

Time Course of SH2B1β Enhancement of Expression of Representative NGF-Regulated Genes Control PC12 or PC12-SH2B1β cells were treated with 100 ng/ml NGF for 0 or 30 min or 1, 2, 4, 6, or 24 h. Relative gene expression levels were determined using QT-PCR and normalized to levels of GAPDH gene expression.

Figure 5

Physiological Functions of uPAR/MMP Activation Cascade Cell surface-localized uPAR, together with cathepsins, binds to pro-uPA and converts inactive pro-uPA to active uPA. The active uPA converts plasminogen to plasmin, and the active plasmin cleaves and thereby activates MMPs. Active MMP has been shown to regulate ECM degradation.

Figure 6

SH2B1β Enhances NGF-Induced uPAR Protein Expression but Does Not Affect the Subcellular Distribution of Cell Surface-Localized uPAR A, PC12 cells stably expressing GFP, GFP-SH2B1β, or GFP-SH2B1β(R555E) were incubated with 100 ng/ml NGF for 0, 4, and 6 h. Proteins in a whole-cell lysate were separated by SDS-PAGE under nonreducing conditions, and uPAR protein levels were determined by immunoblotting with antibody to rat uPAR. Expression levels of GFP-SH2B1β, GFP-SH2B1β(R555E), and actin were determined under reducing conditions by immunoblotting proteins in whole-cell lysates with α-GFP or α-actin, respectively. The molecular weight markers are noted on the left (n = 2). B, PC12 cells stably expressing GFP, GFP-SH2B1β, or GFP-SH2B1β(R555E) plated on Matrigel-coated coverslips were incubated with 100 ng/ml NGF for 0, 7, or 24 h as indicated and fixed with 4% paraformaldehyde but not permeabilized before addition of monoclonal uPAR antibody and antimouse Alexa Fluor 555. Representative confocal images are shown. The inset in I is an image of a different focal plane that focused more on the neurite. The experiment was repeated three times with similar results.

Figure 7

SH2B1β Enhances NGF-Induced MMP3/MMP10 Activity A, Control PC12, PC12-SH2B1β, or PC12-SH2B1β(R555E) cells were treated with 100 ng/ml NGF for 0, 3, 6, 8, and 10 h. Proteins in concentrated conditioned medium were separated in casein-containing, prestained zymogram gels and then developed for 16 h. A representative gel is shown. B, The MMP3/10 activity in A was quantified by band intensity using NIH Image software. C, NGF-induced MMP3/10 activity was quantified for three independent experiments. Means ± sem are shown. MMP3/10 activity levels of the three cell lines are significantly different (P < 0.05, one-tailed, paired Student’s t test) at 10 h, and by 8 h, MMP3/10 activity in PC12-SH2B1β cells was elevated above control cells (P > 0.05).

Figure 8

SH2B1β Enhances NGF-Induced Neurite Invasiveness of PC12 Cells A, Schematic of transwell system used to measure neurite invasiveness. Cells were added onto growth factor-reduced Matrigel on the cis-plane of a transwell insert. Neurite invasiveness was assessed by counting the neurites appearing on the trans-plane of the transwell insert. B, Control PC12 cells or PC12-SH2B1β cells were added to the Matrigel on the cis-plane of a transwell insert, and 100 ng/ml NGF was added to the outer chamber. After 4 d, the cells on the insert were fixed and immunostained with anti-β-tubulin and Alexa Fluor 555. Neurites that traversed to the trans-plane were counted. Means ± sem are shown for n =3.

Figure 9

The siRNA-Mediated Knockdown of Endogenous SH2B1β Inhibits NGF-Dependent Induction of Plaur, Mmp3, and Mmp10 A, Proteins in lysates of PC12 cells stably expressing the control shRNA (shControl) or shRNA targeted against SH2B1 (shSH2B1) were separated by SDS-PAGE and immunoblotted with α-SH2B1 and then anti-α-tubulin as a loading control. B, After incubation in serum-free medium overnight, PC12 cells expressing control shRNA or shRNA targeted against SH2B1 were incubated with or without 50 ng/ml NGF for 6 h. The NGF-dependent induction of mRNA for Plaur, Mmp3, and Mmp10 was quantified via QT-PCR. Gene expression in the presence of NGF was divided by expression in the absence of NGF and then normalized to levels of GAPDH expression in the presence of NGF divided by expression in the absence of NGF. Results were then further normalized to those obtained using shControl cells. Means ± sem are shown for n = 3. *, P < 0.05 using a one-tailed, paired Student’s t test; #, P = 0.06.