Objective To investigate whether hypoxia regulates Notch signaling, and whether Notch plays a role in intervertebral disc cell proliferation. Methods Reverse transcription–polymerase chain reaction and Western blotting were used to measure expression of Notch signaling components in intervertebral disc tissue from mature rats and from human discs. Transfections were performed to determine the effects of hypoxia and Notch on target gene activity. Results Cells of the nucleus pulposus and annulus fibrosus of rat disc tissue expressed components of the Notch signaling pathway. Expression of Notch-2 was higher than that of the other Notch receptors in both the nucleus pulposus and annulus fibrosus. In both tissues, hypoxia increased Notch1 and Notch4 messenger RNA (mRNA) expression. In the annulus fibrosus, mRNA expression of the Notch ligand Jagged1 was induced by hypoxia, while Jagged2 mRNA expression was highly sensitive to hypoxia in both tissues. A Notch signaling inhibitor, L685458, blocked hypoxic induction of the activity of the Notch-responsive luciferase reporters 12xCSL and CBF1. Expression of the Notch target gene Hes1 was induced by hypoxia, while coexpression with the Notch–intracellular domain increased Hes1 promoter activity. Moreover, inhibition of Notch signaling blocked disc cell proliferation. Analysis of human disc tissue showed that there was increased expression of Notch signaling proteins in degenerated discs. Conclusion In intervertebral disc cells, hypoxia promotes expression of Notch signaling proteins. Notch signaling is an important process in the maintenance of disc cell proliferation, and thus offers a therapeutic target for the restoration of cell numbers during degenerative disc disease.

Objective. Since nucleus pulposus cells reside under conditions of hypoxia, we determined if the expression of ANK, a pyrophosphate transporter, is regulated by the hypoxia-inducible factor (HIF) proteins.Methods. Quantitative reverse transcriptionpolymerase chain reaction and Western blot analyses were used to measure ANK expression in nucleus pulposus cells from rats and humans. Transfections were performed to determine the effect of HIF-1/2 on ANK promoter activity.Results. ANK was expressed in embryonic and mature rat discs. Oxygen-dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1␣ and HIF-2␣ resulted in increased ANK expression and up-regulation of promoter activity. HIF-mediated suppression of ANK was validated by measuring promoter activity in HIF-1␤-null embryonic fibroblasts. Under conditions of hypoxia, there was induction of promoter activity in the null cells as compared with the wild-type cells. Overexpression of HIF-1␣ and HIF-2␣ in nucleus pulposus cells resulted in a significant suppression of ANK promoter activity. Since the ANK promoter contains 2 hypoxiaresponsive elements (HREs), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HREs and can suppress promoter activity; in contrast, HIF-2 was required to bind to both HREs in order to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerated state.Conclusion. Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline expression of ANK in the disc serves to prevent mineral formation under physiologic conditions.Within the spine, the vertebrae are separated by a complex tissue, the intervertebral disc. At the periphery of the disc, a ligamentous tissue, the anulus fibrosus, encloses the proteoglycan-rich nucleus pulposus. Although details of the ontology of cells of the adult nucleus pulposus are obscure, it is known that they are derived from the notochord (1), an embryonic tissue with a limited blood supply. The superior and inferior boundaries of the intervertebral disc are formed by the cartilage end plates. A limited number of blood vessels infiltrate the end plates and the outer anulus fibrosus but do not enter the nucleus pulposus (2,3). For this reason, there is considerable support for the view that the nucleus pulposus cells reside in a hypoxic environment (4)(5)(6). Surprisingly, while the disc contains both fibrous proteins and a hydrated extracellular matrix, calcified deposits are absent in the nucleus pulposus and the anulus fibrosus in healthy state.

The objective of our study was to examine the regulation of hypoxic expression of Hsp70 in nucleus pulposus cells and to determine if Hsp70 promoted HIF-1α degradation. Rat nucleus pulposus cells were maintained in culture in either 21% or 1% oxygen. To determine the regulation of Hsp70 expression by Tonicity enhancer binding protein (TonEBP) and HIF-1/2, loss- and gain-of-function experiments and mutational analysis of Hsp70 promoter were performed. Hypoxia increased Hsp70 expression in nucleus pulposus cells. Noteworthy, hypoxia increased TonEBP transactivation and mutation of TonE motifs blocked hypoxic induction of Hsp70 promoter. In contrast, mutation of HRE motifs coupled with loss of function experiments suggested that HIF-1 and HIF-2 suppressed Hsp70 promoter activity and transcription. Interestingly, HIF-α interferes with TonEBP function and suppresses inductive effect of TonEBP on Hsp70 promoter. In terms of Hsp70 function, when treated with Hsp70 transcriptional inhibitor, KNK437, there was an increase in HIF-1α protein stability and transcriptional activity. Likewise, when Hsp70 was overexpressed, the stability of HIF-1α and its transcriptional activity decreased. Hsp70 interacted with HIF-1α under hypoxic conditions and evidenced increased binding when treated with MG132, a proteasomal inhibitor. These results suggest that Hsp70 may promote HIF-1α degradation through proteasomal pathway in nucleus pulposus cells. In hypoxic and hyperosmolar nucleus pulposus cells, Hsp70, TonEBP and HIFs form a regulatory loop. We propose that the positive regulation by TonEBP and negative regulation of Hsp70 by HIF-1 and HIF-2 may serve to maintain Hsp70 levels in these cells, whereas Hsp70 may function in controlling HIF-1α homeostasis.

Objective To study the regulation of expression of β-1,3-glucuronosyltransferase 1 (GlcAT-1), an important regulator of glycosaminoglycan (GAG) synthesis, by Smad3 in nucleus pulposus (NP) cells. Methods GlcAT-1 expression was examined in rat NP and anulus fibrosus (AF) cells treated with transforming growth factor β (TGFβ). The effects of Smad signaling and Smad suppression on GlcAT-1 were examined in rat NP cells. GlcAT-1 expression was analyzed in the discs of Smad3-null mice and in degenerated human NP tissue. Results TGFβ increased the expression of GlcAT-1 in rat NP but not rat AF cells. Suppression of GlcAT-1 promoter activity was evident with dominant-negative ALK-5 (DN-ALK-5). Cotransfection with Smad3 strongly induced promoter activity independent of TGFβ. Bioinformatics analysis indicated the presence of several Smad binding sites in the promoter; deletion analysis showed that the region between −274 and −123 bp was required for Smad3 response. DN-Smad3, Smad 3 small interfering RNA, and Smad7 strongly suppressed basal as well as TGFβ-induced promoter activity. Induction of promoter activity by Smad3 was significantly blocked by DN-Smad3; Smad7 had a very small effect. Lentiviral transduction of NP cells with short hairpin RNA Smad3 resulted in a decrease in GlcAT-1 expression and accumulation of GAG. Compared to wild-type mice, significantly lower expression of GlcAT-1 was seen in the discs of Smad3-null mice. Analysis of degenerated human NP tissue specimens showed no positive correlation between GlcAT-1 and TGFβ expression. Moreover, isolated cells from degenerated human tissue showed a lack of induction of GlcAT-1 expression following TGFβ treatment, suggesting an altered response. Conclusion Our findings demonstrate that in healthy NP cells, the TGFβ–Smad3 axis serves as a regulator of GlcAT-1 expression. However, an altered responsiveness to TGFβ during disc degeneration may compromise GAG synthesis.

The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. A high-yield, scalable, cGMP-compliant downstream process for the stabilized, soluble, native-like S protein ectodomain is necessary to meet the extensive material requirements for ongoing research and development. As of June 2021, S proteins have exclusively been purified using difficult-to-scale, low-yield methodologies such as affinity and size-exclusion chromatography. Herein we present the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44. A high-throughput resin screen on the Tecan Freedom EVO200 automated bioprocess workstation led to identification of ion exchange resins as viable purification steps. The chromatographic unit operations along with industry-standard methodologies for viral clearances, low pH treatment and 20 nm filtration, were assessed for feasibility. The developed process was applied to purify HexaPro from a CHO-DG44 stable pool harvest and yielded the highest yet reported amount of pure S protein. Our results demonstrate that commercially available chromatography resins are suitable for cGMP manufacturing of SARS-CoV-2 Spike protein constructs. We anticipate our results will provide a blueprint for worldwide biopharmaceutical production laboratories, as well as a starting point for process intensification.