1. Hypoxia regulates sumoylation pathways in intervertebral disc cells: implications for hypoxic adaptations 
F. Wang, F. Cai, R. Shi, J.-N. Wei, X.-T. Wu 
Osteoarthritis and Cartilage 
Volume 24, Issue 6, Pages (June 2016)
DOI: /j.joca
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2. Fig. 1
Monolayer cultures of rat lumber IVD cells. A, B: primary NP cells (A) and AF cells (B) migrated out of the partially digested explant after 3–7 days cultures under normoxia (original magnification ×40). The primary AF cells proliferated faster than NP cells and tended to form large and dense cell clusters (B). C, D: the sub-cultured NP (C) and AF cells (D) at passage 1 (P1). The cytoplasm-vacuolated notochordal cells (arrow) were seen in the primary and sub-cultured NP cells, which gradually disappeared by P3 (original magnification ×100).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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3. Fig. 2
Expression of SUMO and SUMO pathway elements in neonatal rat IVD cells. Immunohistochemical staining of SUMO-1, SUMO-2/3, SAE1, SAE2, UBC9, and SENP1 in the coronal section of neonate rat IVD. Both the SUMO molecules and sumoylation enzymes were expressed in the cells of NP, AF, and CEP, mainly in the nuclei (original magnification ×400).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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4. Fig. 3
Expression of SUMO and SUMO pathway elements in adult rat disc cells. A: immunocytofluorescent staining of SUMO and SUMO pathway elements in the primary NP cells isolated from adult (8–12-week old) rat IVD. SUMO-1 was exclusively expressed in the nuclei, while SUMO-2/3, SAE1, SAE2, UBC9, and SENP1 were detected both in the nuclei and cytoplasm, but most prominent in the nuclei (original magnification ×100, negative controls and expressions in AF cells were not shown). B–D: immunohistochemical staining of SUMO-1 (B), SUMO-2/3 (C), SAE1 (D) in adult (10-week old) rat IVD. The predominant nuclear expression of SUMO molecules and pathway elements were also evidenced in the NP, AF, and CEP of adult IVD (original magnification ×400).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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5. Fig. 4
Hypoxic regulation of SUMO molecules in IVD cells. A–C: mRNA expression of SUMO molecules under hypoxia (Hx) for 0 h (normoxia, Nx) to 24 h. In NP cells the SUMO-1 transcription was significantly increased after exposure to hypoxia for 4 h (0 h vs 4 h P < 0.0001; 0 h vs 12 h P = 0.23; 0 h vs 24 h P = 0.07), whereas in AF cells the transcription of SUMO-1 showed increase after hypoxic cultures for 12 h (0 h vs 4 h P = 0.54; 0 h vs 12 h P < 0.0001; 0 h vs 24 h P = 0.93). In NP cells the SUMO-2 and SUMO-3 transcription was significantly increased after exposure to hypoxia for 4 h (0 h vs 4 h P <  for SUMO-2 and SUMO-3; 0 h vs 12 h P = 0.33 for SUMO-2 and P = 0.45 for SUMO-3; 0 h vs 24 h P = 0.99 for SUMO-2 and SUMO-3), while AF cells decreased the mRNA level of SUMO-2/3 in hypoxia (0 h vs 4 h P = 0.61 for SUMO-2 and P = 0.68 for SUMO-3; 0 h vs 12 h P = 0.003 for SUMO-2 and P = 0.002 for SUMO-3; 0 h vs 24 h P = 0.002 for SUMO-2 and P = 0.007 for SUMO-3). D, E: protein expression of SUMO molecules in NP and AF cells. In rat disc cells SUMO-1 was detected at 12 kD and 80 kD (D), while SUMO-2/3 was detected at 11 kD (E). F: RanGAP1 was detected in NP and AF cells at ∼80 kD under both normoxia and hypoxia (for 24 h), suggesting the 80 kD SUMO-1 was probably the RanGAP1–SUMO-1 complexity. G, H: hypoxic expression of the conjugated SUMO-1 and free SUMO-2/3. In NP cells the conjugated SUMO-1 was significantly increased after maintained in hypoxia for 4–12 h (0 h vs 4 h and 12 h P < 0.0001; 0 h vs 24 h P = 0.69), whereas the conjugation of SUMO-1 was elevated in the AF cells under hypoxia for 12–24 h (0 h vs 4 h P = 0.08; 0 h vs 12 h and 24 h P < 0.0001). NP cells increased the content of non-conjugated SUMO-2/3 after maintained in low oxygen tensions for 4 h (0 h vs 4 h P < 0.0001; 0 h vs 12 h P = 0.01; 0 h vs 24 h P = 0.63), the free SUMO-2/3 within AF cells was reduced after hypoxic culture for 4–12 h (0 h vs 4 h P < 0.0001; 0 h vs 12 h P = 0.01), which returned to the level of normoxic cultures by 24 h (0 h vs 24 h P = 0.06). * = statistically significant difference and NS = not significant.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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6. Fig. 5
Hypoxic regulation of sumoylation enzymes in IVD cells. A–C: mRNA expression of sumoylation enzymes under hypoxia (Hx) for 0 h (normoxia, Nx) to 24 h. In NP cells the transcription of SAE1 showed transient decrease after maintained in hypoxia for 4–12 h (0 h vs 4 h P = 0.0005; 0 h vs 12 h P < 0.0001; 0 h vs 24 h P = 0.13), whereas the expression of SAE1 was decreased in AF cells under hypoxia (0 h vs 4 h P = 0.71; 0 h vs 12 h and 24 h P < 0.0001). While SAE2 was lowered by hypoxia within AF cells (0 h vs 4 h P = 0.79; 0 h vs 12 h and 24 h P < 0.0001), NP cells transiently elevated the transcription of SAE2 after exposure to hypoxia for 4 h (0 h vs 4 h and 24 h P < 0.0001; 0 h vs 12 h P = 0.13). A hypoxia-induced transient increase of UBC9 was also noted in NP cells (0 h vs 4 h P < 0.0001; 0 h vs 12 h P = 0.86; 0 h vs 24 h P = 0.99), while AF cells decreased the expression of UBC9 under low oxygen tensions (0 h vs 4 h P = 0.84; 0 h vs 12 h P = 0.021; 0 h vs 24 h P = 0.019). In both NP (0 h vs 4–24 h P < 0.0001) and AF cells (0 h vs 4 h P = 0.99; 0 h vs 12 h P = 0.003; 0 h vs 24 h P < 0.0001), the transcription of SENP1 showed steady increase under hypoxic cultures. E, F: the protein expression of sumoylation pathways was in line with the hypoxia-induced transcriptional change in NP and AF cells. * = statistically significant difference and NS = not significant.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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7. Fig. 6
SENP1 regulates the expression and transactivation of HIF-1α in NP cells. A: silencing of SENP1 by the SENP1 specific shRNA with LV transfection. The immunofluorescence analysis showed high (>95%) transduction efficiency of the plasmid encoding control shRNA (LV-sh Ctr) or SENP1 specific interfering shRNA (LV-sh SENP1) in NP cells (original magnification ×40). As compared to the LV-sh Ctr group, the transcription of SENP1 (A2) was significantly interfered by SENP1 specific shRNA (P < 0.0001), which was further confirmed by the suppressed expression of SENP1 on western blotting (A3). B: hypoxic regulation of HIF-1α expression in NP cells. HIF-1α was constitutively expressed within NP under both normoxia and hypoxia (B1). Hypoxia stabilized and enhanced (2–3 folds) the expression of HIF-1α in the control (P = 0.006) and LV-sh Ctr (P = 0.002) group (B2), whereas the NP cells with suppressed SENP1 (P =  ) reduced the protein level of HIF-1α under hypoxia. Similarly, while the transcription of HIF-1α (B3) was enhanced by hypoxia (for 24 h) in the normal (P < 0.0001) and the control shRNA transfected (P < 0.0001) NP cells, silencing SENP1 reduced the mRNA expression of HIF-1α under low oxygen tensions (P = 0.002). C: hypoxic regulation of HIF-1α transactivation in NP cells. Hypoxic cultures for 24 h increased the mRNA expression of GLUT-1 and VEGF within the normal (for GLUT-1 P = 0.001 and VEGF P = 0.0001) and LV-sh Ctr (for GLUT-1 P = 0.001 and VEGF P < 0.0001) group, whereas the NP cells with silenced SENP1 decreased the transactivation of HIF-1α under hypoxia (for GLUT-1 P = 0.006 and VEGF P = 0.016). D: hypoxic regulation of PDK1 in disc cells. The expression of PDK1 was increased by hypoxia (24 h) in NP (P = 0.0013) and AF (P =  ) cells (D1 and D2). After SENP1 was silenced by LV-sh SENP1 transfection (D3 and D4), unlike the control (P = 0.003) and LV-sh Ctr (P = 0.007) group, the NP cells with suppressed SENP1 showed no elevation of PDK1 under hypoxia (P = 0.39). E: Hoechst33342/PI staining and flow cytometry analysis of apoptosis. As compared to the normoxic cultures, no significant elevation of apoptosis was induced by hypoxia in the NP cells transfected with LV-sh Ctr (P = 0.55) or LV-sh SENP1 (P = 0.73).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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8. Fig. 7
Hypoxic regulation of IVD cells viability and proliferation. A, B: CCK-8 array of disc cell proliferation under hypoxia (Hx) and normoxia (Nx). No significant change of growth kinetics was induced by hypoxia in both NP (A, for 0 h P = 0.93, 24 h P = 0.58; 48 h P = 0.39; 72 h P = 0.49) and AF cells (B, for 0 h P = 0.35, 24 h P = 0.57; 48 h P = 0.92; 72 h P = 0.54). C: representative of SA-β-gal staining of NP and AF cells maintained in normoxia and hypoxia for 24 h. Cells with increased SA-β-gal activity was stained deep blue (C) and hypoxia induced no significant elevation of cellular senescence (D) in NP (0 h vs 12 h and 48 h P = 0.99, 0 h vs 24 h P = 0.52) or AF cells (0 h vs 12 h and 48 h P = 0.99, 0 h vs 24 h P = 0.97). E: representative of flow cytometric analysis of apoptosis rate and cell cycle distribution in the AF cells (NP cells not shown) cultured under hypoxia for 0–48 h. No significant apoptosis (F) was induced by hypoxia in NP (0 h vs 12–48 h P = 0.99) or AF cells (0 h vs 12 h and 24 h P = 0.99; 0 h vs 48 h P = 0.96). Cells in the G1-phase (G) was not increased by hypoxia in NP (0 h vs 24 h P = 0.99, 0 h vs 48 h P = 0.60) or AF cells (0 h vs 24 h P = 0.97, 0 h vs 48 h P = 0.96), nor the ratio of S-phase was decreased in the NP (0 h vs 24 h P = 0.31, 0 h vs 48 h P = 0.88) or AF cells (0 h vs 24 h P = 0.96, 0 h vs 48 h P = 0.61) exposed to low oxygen tensions. NS = not significant.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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9. Fig. 8
Schematic illustrating the hypoxic regulation of sumoylation pathways in IVD cells. A: the avascular nature of IVD. Since blood supply only reaches to the superficial region of CEP and the outer surface of AF, oxygen tension undergoes significant reduction from disc surface to its inner part. B: hypoxia differently regulates sumoylation pathways in NP and AF cells. In response to oxygen deficiency, the global balance of SUMO conjugations might shift to the de-sumoylation end, especially in the cells of AF. Although hypoxia enhances SUMO-1 conjugation in both NP and AF cells, more dynamic regulation of SUMO-2/3 conjugation and de-conjugation might occur in the cells of AF. The distinct sumoylation manner within NP and AF cells might differently modulate various hypoxia-responsive gene products, such as HIF and GLUT, which are sumoylation substrates and mediate cell survival and metabolism within the avascular IVD.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions