PAK1 Promotes the Proliferation and Inhibits Apoptosis of Human Spermatogonial Stem Cells via PDK1/KDR/ZNF367 and ERK1/2 and AKT Pathways

Abstract

Spermatogonial stem cells (SSCs) have significant applications in reproductive and regenerative medicine. However, nothing is known about genes in mediating human SSCs. Here we have explored for the first time the function and mechanism of P21-activated kinase 1 (PAK1) in regulating the proliferation and apoptosis of the human SSC line. PAK1 level was upregulated by epidermal growth factor (EGF), but not glial cell line-derived neurotrophic factor (GDNF) or fibroblast growth factor 2 (FGF2). PAK1 promoted proliferation and DNA synthesis of the human SSC line, whereas PAK1 suppressed its apoptosis in vitro and in vivo. RNA sequencing identified that PDK1, ZNF367, and KDR levels were downregulated by PAK1 knockdown. Immunoprecipitation and Western blots demonstrated that PAK1 interacted with PDK1. PDK1 and KDR levels were decreased by ZNF367-small interfering RNAs (siRNAs). The proliferation of the human SSC line was reduced by PDK1-, KDR-, and ZNF367-siRNAs, whereas its apoptosis was enhanced by these siRNAs. The levels of phos-ERK1/2, phos-AKT, and cyclin A were decreased by PAK1-siRNAs. Tissue arrays showed that PAK1 level was low in non-obstructive azoospermia patients. Collectively, PAK1 was identified as the first molecule that controls proliferation and apoptosis of the human SSC line through PDK1/KDR/ZNF367 and the ERK1/2 and AKT pathways. This study provides data on novel gene regulation and networks underlying the fate of human SSCs, and it offers new molecular targets for human SSCs in translational medicine.

Keywords: ERK1/2 and AKT pathways; PAK1; PDK1/KDR/ZNF367; apoptosis; human spermatogonial stem cells; proliferation.

Figure 1

The Effect of EGF, FGF2, and GDNF on PAK1, EGFR Presence in the Human SSC Line, and the Expression of PAK1 in Human Primary SSCs (A) Real-time PCR displayed mRNA changes of PAK1 by growth factors EGF, FGF2, and GDNF in the human SSC line. (B and C) Western blots showed protein changes of PAK1 (B) and its relative level (C) by these growth factors mentioned above in the human SSC line. Culture medium without EGF, FGF2, or GDNF was used as the control. (D and E) Western blots demonstrated the protein changes of PAK (D) or its relative level (E) by EGF, GDNF, or FGF2 or EGF + GDNF + FGF2 in the human SSC line. *Statistically significant differences (p < 0.05) between the individual growth factor, EGF + FGF2 + GDNF-treated cells, and the control (A, C, and E). (F and G) RT-PCR and immunocytochemistry showed the expression of EGFR mRNA (F) and EGFR protein (G) in the human SSC line. Scale bar, 10 μm (G). (H) Immunohistochemistry revealed cellular localization of PAK1 in human spermatogonia (arrows), but not pachytene spermatocytes (asterisks) or round spermatids (arrow heads). (I) Replacement of anti-PAK1 with isotype IgG was used as a negative control. Scale bars, 5 μm (H and I).

Figure 2

The Influence of PAK1 Knockdown on the Proliferation, DNA Synthesis, and Apoptosis of the Human SSC Line (A) Real-time PCR showed mRNA changes of PAK1 by PAK1-siRNA 1, 2, and 3 in the human SSC line. (B) Western blots revealed the protein changes of PAK1 by PAK1-siRNA 1, 2, and 3 in the human SSC line. (C) CCK-8 assay demonstrated the proliferation of the human SSC line after transfection of PAK1-siRNA 1 and 2. (D) Western blots displayed the changes of PCNA protein by PAK1-siRNA 1 and 2 in human SSCs. (E–G) BrdU incorporation assay showed the percentages of BrdU-positive cells affected by control siRNA (E and G) and PAK1-siRNA 2 (F and G) in the human SSC line. Scale bars, 20 μm (E and F). (H and I) Annexin V/PI staining and flow cytometry displayed the percentages of early (I, left panel) and late (I, right panel) apoptosis in the human SSC line affected by PAK1-siRNA 2 (H, right panel) and control siRNA (H, left panel). *Statistically significant differences (p < 0.05) between PAK1-siRNA -treated cells and the control siRNA (A–D, G, and I).

Figure 3

PAK1-siRNA 2 Knockdown Led to the Reduction of Proliferation and the Increase of Apoptosis in the Recipient Mice Grafted with the Human SSC Line (A–F) Immunohistochemistry showed the expression of UCHL1- (A), PLZF- (B), PCNA- (C), Ki67- (D), and TUNEL- (E) positive cells and their percentages (F) in the recipient nude mice grafted with the human SSC line by PAK1-siRNA 2 or control siRNA transfection. Scale bars, 10 μm (A–E). *Statistically significant differences (p < 0.05) between PAK1-siRNA 2-treated cells and the control siRNA. Notes in (F): the percentage of TUNEL-positive cells was calculated by TUNEL-positive positive cells from all cells within the seminiferous tubule. The percentage of UCHL1-, PLZF-, PCNA-, and Ki67-positive cells were counted by the positive cells from the cells along the basement membrane. At least 3 integrated seminiferous tubules were randomly selected in each testicular section, and at least 5 sections were counted.

Figure 4

Identification of PDK1, KDR, and ZNF367 as the Targets for PAK1 in Human SSCs (A and B) Electropherogram showed the concentrations and nucleotides (nt) of RNA isolated from the human SSC line with the control siRNA (A) and PAK1-siRNA 2 (B). (C and D) Scatterplots (C) and hierarchical clustering (D) illustrated the differentially expressed genes (DEGs) between PAK1-siRNA 2 and control siRNA. Red dots and green dots in (C) represented upregulated and downregulated genes, respectively. (E) Real-time PCR revealed the changes of PDK1, KDR, ZNF367, GPX3, OIP5, THAP10, DBP, and TET1 mRNA by PAK1-siRNA 2 compared to the control siRNA. *Statistically significant differences (p < 0.05) between PAK1-siRNA 2-treated cells and the control siRNA.

Figure 5

The Influence of PDK1 Knockdown on the Proliferation, DNA Synthesis, and Apoptosis of the Human SSC Line (A) Western blots showed protein changes of PDK1 by PAK1-siRNA 1 and 2 in the human SSC line. *Statistically significant differences (p < 0.05) between PAK1-siRNA 1- and 2-treated cells and the control siRNA. (B) Immunoprecipitation assay and Western blots demonstrated the binding of anti-PAK1 to PDK1 in the human SSC line. 1, IgG; 2, anti-PAK1. (C) Real-time PCR revealed the mRNA changes of PDK1 by PDK1-siRNA 1, 2, and 3 in the human SSC line. (D) Western blots revealed the protein changes of PDK1 by PDK1-siRNA 1, 2, and 3 in the human SSC line. (E) CCK-8 assay showed the proliferation of the human SSC line after transfection of PDK1-siRNA 1 and 3. (F–H) EDU incorporation assay demonstrated the percentages of EDU-positive cells affected by control siRNA (F and H) and PDK1-siRNA 3 (G and H) in the human SSC line. Scale bars, 20 μm (F and G). (I and J) Annexin V/PI staining and flow cytometry demonstrated the percentages of early (J, left panel) and late apoptosis (J, right panel) in the human SSC line treated with PDK1-siRNA 3 (I, right panel) and control siRNA (I, left panel). *Statistically significant differences (p < 0.05) between PDK1-siRNA-treated cells and the control siRNA (C–E, H, and J).

Figure 6

The Role of KDR Silencing on the Proliferation, DNA Synthesis, and Apoptosis of the Human SSC Line (A and B) Western blots showed protein changes of KDR (A) and its relative level (B) by PAK1-siRNA 1 and 2 in the human SSC line. *Statistically significant differences (p < 0.05) between PAK1-siRNA 1- and 2-treated cells and the control siRNA. (C) Real-time PCR showed mRNA changes of KDR by KDR-siRNA 1, 2, and 3 in the human SSC line. (D and E) Western blots demonstrated protein changes of KDR (D) and its relative level (E) by KDR-siRNA 1, 2, and 3 in the human SSC line. (F) CCK-8 assay showed the proliferation of the human SSC line after transfection of KDR-siRNA 3 and KDR-siRNA 1. (G–I) EDU incorporation assay illustrated the percentages of EDU-positive cells affected by the control siRNA (G and I) and KDR-siRNA 3 (H and I) in the human SSC line. Scale bars, 10 μm (G and H). (J–L) Annexin V/PI staining and flow cytometry demonstrated the percentages of early (K) and late (L) apoptosis in the human SSC line treated with KDR-siRNA 3 (J, right panel) and control siRNA (J, left panel). *Statistically significant differences (p < 0.05) between KDR-siRNA-treated cells and the control siRNA (C, E, F, I, K, and L).

Figure 7

The Influence of PAK1-siRNAs on the Levels of phos-ERK1/2, phos-AKT, P85, and Cell Cycle Proteins in the Human SSC Line (A–E) Western blots showed the level changes of phos-ERK1/2 (A and C), phos-AKT (B and C), P85 (B and C), cyclin A (D and E), cyclin B1 (D and E), cyclin D1 (D and E), and CDK2 (D and E) in the human SSC line with PAK1-siRNA 1 and 2 and control siRNA. ERK2, AKT, and ACTB were used as controls of loading proteins. *Statistically significant differences (p < 0.05) between PAK1-siRNA 1- and 2-treated cells and the control siRNA.

Figure 8

The Effectiveness of ZNF367 Silencing on the Proliferation, DNA Synthesis, and Apoptosis of the Human SSC Line (A and B) Western blots showed protein changes of ZNF367 (A) and its relative level (B) by PAK1-siRNA 1 and 2 in the human SSC line. *Statistically significant differences (p < 0.05) between PAK1-siRNA 1- and 2-treated cells and the control siRNA. (C) Real-time PCR revealed mRNA changes of ZNF367 by ZNF367-siRNA 1, 2, and 3 in the human SSC line. (D and E) Western blots showed protein changes of ZNF367 (D) and its relative level (E) by ZNF367-siRNA 1, 2, and 3 in the human SSC line. (F and G) Real-time PCR revealed mRNA changes of KDR (F) and PDK1 (G) by ZNF367-siRNA 1 and 2 in the human SSC line. (H) CCK-8 assay showed the proliferation of the human SSC line after transfection of ZNF367-siRNA 1 and 2. (I–K) EDU incorporation assay illustrated the percentages of EDU-positive cells affected by control siRNA (I and K) and ZNF367-siRNA 1 (J and K) in the human SSC line. Scale bars, 20 μm (I and J). (L–N) Annexin V/PI staining and flow cytometry demonstrated the percentages of early (M) and late (N) apoptosis in the human SSC line treated with ZNF367-siRNA 1 (L, right panel) and control siRNA (L, left panel). *Statistically significant differences (p < 0.05) between ZNF367-siRNA-treated cells and the control siRNA (C, E–H, K, M, and N).

Figure 9

The Expression of PAK1 in Male Germ Cells and the Testis of OA Patients and Certain Subtypes of NOA Patients (A–C) Real-time PCR and Western blots revealed the transcripts of PAK1 (A) and PAK1 protein (B and C) in male germ cells of NOA patients and OA patients. (D and E) Tissue arrays showed the levels of PAK1 protein (D) and its relative level (E) in MA at spermatogonia, spermatocytes, or spermatids, HS patients, and OA patients. Specific immunostaining of PAK1 in human spermatogonia (arrows) was shown in an OA patient. *Statistically significant differences (p < 0.05) between NOA patients and OA patients (C and E). The quantification of PAK1 immunostaining was determined by calculating the number scores of PAK1-positive cells and the intensity scores of staining.

Figure 10

Schematic Diagram Illustrates and Summarizes the Role and Signaling Pathway of PAK1 in Regulating Human SSCs PAK1 is mediated by EGF, but not by GDNF or FGF2; PDK1, ZNF367, and KDR are the targets of PAK1. PAK1 binds to PDK1, and ZNF367 regulates PDK1 and KDR. PAK1 enhances the phosphorylation (phos-) of ERK1/2 and AKT, which facilitates directly or indirectly the entrance of phos-ERK1/2 and phos-AKT from cytoplasm to nuclei. PAK1 increases cyclin A level and stimulates human SSCs to enter S phase for DNA synthesis and cellular proliferation. Solid arrows, promote directly; dotted arrows, stimulate directly or indirectly; P in a circle, phosphorylate.