The Function And Regulative Mechanism Of Carbonic Anhydrase 12 In Lumbar Intervertebral Disc Nucleus Pulposus Degeneration

Part I Evaluation of novel nucleus pulposus(NP) markers in degenerative discsObjective:To evaluate 12 new candidate NP markers include carbonic anhydrase 12(CA12) in degenerative disc disease in a Chinese population.Methods:Specimens of NP were collected from 81 patients undergoing spine surgeries. These patients were grouped into the degenerated disc group and the control group. Lumbar spine MRI, and H&E staining and safranin O staining of sections of NP tissues were conducted to evaluate the severity of degeneration in all samples. Quantitative real-time PCR was performed to investigate the levels of mRNA expression of these genes, as well as those of aggrecan, type Ⅱ collagen and SRY-box 9(SOX9). We also used evaluated the expression of CA12 by immunohistochemistry and Western Blot.Results. We demonstrated that CA12 and other 11 candidates showed significant differential expression in degenerated discs. Changes in the expression of these genes were determined to be risk factors in degenerative disc diseases. The expression of CA12, neurochondrin (NCDN), keratin 8 (KRT8), and matrix Gla protein (MGP) even showed significant changes among subgroups of patients with degenerative disc disease stratified according to the Pfirrmann grading system. CA12 also showed differential expression in protein level.Conclusions. Overall, we demonstrated the clinical utility of CA12 and 11 novel NP markers for degenerative disc disease. Among them, the expression of CA12、NCDN, KRT8 and MGP may indicate the severity of disc degeneration.Part II The function and regulative mechanism of CA12 in NP cellsObjective. To elucidate how CA12 was regulated and its exact function in disc degeneration.Methods. The expression of HIF-1α and HIF-2a were tested in 81 human degenerated NP samples by real-time RT-PCR, immunohistochemistry and Western Blot. Rat NP cells were cultured under hypoxia to observe the hypoxia-induced expression of CA12. Rat NP cells were treated with HIF-1α siRNA or PHD inhibitor, DMOG to evaluate the role of PHD/HIF-1 in regulating CA12 expression. Rat NP cells were treated with CA12 siRNA and/or NaOH to determine the function of CA12 and the role of pH.Results. Expression of CA12 can be sharply induced in hypoxic condition by about 30-fold. Expression of HIF-la, but not HIF-2a was also decreased in degenerated human NP samples and was positively correlated with that of CA12. Knock-down of HIF-la in hypoxia can lead to reduced expression of CA12 in both mRNA and protein levels. Increased expression of HIF-1αand CA12 were seen when treated with DMOG. Anabolic proteins were significantly suppressed after CA12 was knocked down while catabolic enzymes remained unchanged. And elevation of pH may lead to up-regulation of type II collagen and SOX9.Conclusion. CA12 was down-regulated in degenerated NP and this process may be regulated by PHD/HIF-1 axis. The suppressed expression of CA12 may lead to decreased ECM synthesis and contribute to the progression of degenerative disc disease by regulating intracellular pH.Part III The role of PHD/HIF-1/CA12 in single intervertebral disc ex vivo modelObjective:to confirm the anabolism role of PHD/HIF-1/CA12 axis in ex vivo model.Methods:The single intervertebral disc ex vivo model was established. We used DMOG and/or CA inhibitor ACTZ in the treatment of the ex vivo model. H&E staining and Safranin O were used to evaluate the severity of degeneration. Quantitative real-time PCR were used to test the mRNA levels of aggrecan. type II collagen、type I collagen、SOX9、CA12 and HIF-la. Western Blot was used to evaluate the protein levels of CA12 and HIF-la.Results:The extracellular matrix (ECM) of NP was increased in the DMOG group and the expression of aggrecan、type Ⅱ collagen and SOX9 were also increased. While DMOG+ACTZ group showed decreased ECM and expression of aggrecan、type II collagen and SOX9. Additionally, treatment of DMOG up-regulated the mRNA and protein expression of CA12 and HIF-la.Conclusion:In ex vivo model, inhibition of PHD could enhance anabolism of NP while inhibition of CA could attenuate this effection, suggesting PHD/HIF-1/CA12 may play a role in NP anabolism.