LncRNA LINC00324 is upregulated in intervertebral disk degeneration and upregulates FasL in nucleus pulposus cells

Background

It has been reported that long intergenic non-protein-coding RNA 324 (LINC00324) promotes liver cancer by upregulating Fas ligand (FasL), which is a major player in intervertebral disk degeneration (IDD), indicating the involvement of LINC00324 in IDD. This study was carried out to investigate the interaction between LINC00324 and FasL in IDD.

Methods

Plasma samples were collected from both IDD (n?=?60) and healthy controls (n?=?60). The expression of LINC00324 and FasL in plasma was determined by RT-qPCR. The interactions between LINC00324 and FasL in nucleus pulposus (NP) cells were analyzed by overexpression experiments.

Results

LINC00324 and FasL were upregulated in IDD patients, and they were positively correlated. After treatment, the expression levels of FasL and LINC00324 were significantly decreased. In NP cells, overexpression of LINC00324 increased the expression of FasL at both mRNA and protein levels, while overexpression of FasL did not affect the expression of LINC00324.

Conclusion

LINC00324 may upregulate FasL in IDD to promote disease progression.

     

Increased expression of transglutaminase 2 drives glycolytic metabolism in renal carcinoma cells

Transglutaminase 2 (TGase 2) expression and glycolysis are increased in most renal cell carcinoma (RCC) cell lines compared to the HEK293 kidney cell line. Although increased glycolysis and altered tricarboxylic acid cycle are common in RCC, the detailed mechanism by which this phenomenon occurs remains to be elucidated. In the present study, TGase 2 siRNA treatment lowered glucose consumption and lactate levels by about 20–30 % in RCC cells; conversely, high expression of TGase 2 increased glucose consumption and lactate production together with decreased mitochondrial aconitase (Aco 2) levels. In addition, TGase 2 siRNA increased mitochondrial membrane potential and ATP levels by about 20–30 % and restored Aco 2 levels in RCC cells. Similarly, Aco 2 levels and ATP production decreased significantly upon TGase 2 overexpression in HEK293 cells. Therefore, TGase 2 leads to depletion of Aco 2, which promotes glycolytic metabolism in RCC cells.     

Environmental regulation of notochordal gene expression in nucleus pulposus cells

Cells of the nucleus pulposus (NP) in the intervertebral disc are derived directly from the embryonic notochord. In humans, a shift in NP cell population coincides with the beginning of age‐related changes in the extracellular matrix that can lead to spinal disorders. To begin identifying the bases of these changes, the manner by which relevant environmental factors impact cell function must be understood. This study investigated the roles of biochemical, nutritional, and physical factors in regulating immature NP cells. Specifically, we examined cell morphology, attachment, proliferation, and expression of genes associated with the notochord and immature NP (Sox9, CD24, and type IIA procollagen). Primary cells isolated from rat caudal discs were exposed to different media formulations and physical culture configurations either in 21% (ambient) or 2% (hypoxic) O₂. As expected, cells in alginate beads retained a vacuolated morphology similar to chordocytes, with little change in gene expression. Interestingly, NP tissues not enzymatically digested were more profoundly influenced by oxygen. In monolayer, α‐MEM preserved vacuolated morphology, produced the highest efficiency of attachment, and best maintained gene expression. DMEM and Opti‐MEM cultures resulted in high levels of proliferation, but these appeared to involve small non‐vacuolated cells. Gene expression patterns for cells in DMEM monolayer cultures were consistent with chondrocyte de‐differentiation, with the response being delayed by hypoxia. Overall, results indicate that certain environmental conditions induce cellular changes that compromise the notochordal phenotype in immature NP. These results form the foundation on which the mechanisms of such changes can be elucidated. J. Cell. Physiol. 220: 698–705, 2009. © 2009 Wiley‐Liss, Inc.     

Hypoxia regulates avian cardiac Arnt and HIF-1alpha mRNA expression

The aryl hydrocarbon receptor nuclear translocator (Arnt) and hypoxia-inducible factor (HIF)-1alpha mediate cellular responses to hypoxia. We investigated the ability of hypoxia to regulate Arnt and HIF-1alpha mRNA in the heart in vivo. We cloned avian Arnt, developed an in vivo model of chronic cardiac hypoxia, and measured expression of cardiac Arnt and HIF-1alpha mRNA by quantitative RT-PCR. Chronic hypoxic exposure (24 h to 15% O(2)) of day 9 chick embryos resulted in a 30-fold increase in covalent binding of (3)H-misonidazole, a hypoxic tissue marker, to cardiac tissue, and a 2-fold induction of cardiac inducible nitric oxide synthase mRNA, compared to normoxic controls. In this same model, cardiac Arnt mRNA expression decreased by 35%, while HIF-1alpha mRNA expression increased 400%. These data suggest that regulation of Arnt and HIF-1alpha mRNA expression may contribute to the physiological responses of the heart during prolonged hypoxia.     

HIF-2alpha regulates glyceraldehyde-3-phosphate dehydrogenase expression in endothelial cells

Endothelial cells (EC) express both hypoxia inducible factor-1alpha (HIF-1alpha) and -2alpha (HIF-2alpha), yet their roles in the EC hypoxic response are unclear. Hypoxia upregulates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EC through a 5' hypoxic regulatory element (HRE). We compared the upregulation of GAPDH in human lung microvascular EC to that in hep3B cells, another cell type known to express both HIF-1alpha and HIF-2alpha. GAPDH mRNA increased to a lesser extent in hypoxic hep3B cells than in EC, yet upregulation occurred through the same HRE that was active in EC. HIF-1alpha protein induction in response to hypoxia was similar in both cell types. In contrast, HIF-2alpha protein levels were upregulated to a greater extent and for a longer period of time by hypoxia in EC than in hep3B cells. Correspondingly, electrophoretic mobility supershift assays showed that, in EC, there was preferential binding of HIF-2alpha to the GAPDH HRE while, in hep3B cells, there was binding of both HIF-1alpha and HIF-2alpha. The preferential binding of HIF-2alpha to the GAPDH HRE in EC may account for their higher level of induction of GAPDH. These findings suggest that cell-specific patterns of HIF-1alpha and HIF-2alpha expression lead to cell-specific gene upregulation during hypoxia.     

Expression of acid-sensing ion channels in nucleus pulposus cells of the human intervertebral disk is regulated by non-steroid anti-inflammatory drugs

Non-steroid anti-inflammatory drugs （NSAIDs） are general- ly used in the treatment of inflammation and pain through cyclooxygenase （COX） inhibition. Mounting evidence has indicated additional COX-independent targets for NSAIDs including acid-sensing ion channels （ASICs） la and 3. However, detailed function and mechanism of ASICs still remain largely elusive. In this study, the impact of NSAIDs on ASICs in nucleus puiposus cells of the human interverte- bral disk was investigated. Nucleus pulposus cells were iso- lated and cultured from protruded disk tissues of 40 patients. It was shown that ASICla and ASIC3 were expressed and functional in these cells by analyzing proton- gated currents after ASIC inhibition. We further investi- gated the neuroprotective capacity of ibuprofen （a COX in- hibitor）, psaimotoxin-1 （PcTX1, a tarantula toxin specific for homomeric ASICla）, and amiloride （a classic inhibitor of the epithelial sodium channel ENaC/DEG family to which ASICs belong）. PcTXl-containing venom has been shown to be comparable with amiloride in its neuroprotective features in rodent models of ischemia. Taken together, our data showed that amiloride, PcTX1, and ibuprofen decreased ASIC protein expression and thereby exerted protective effects from ASIC inhibition-mediated cell damage.     

Oligomycin inhibits HIF-1alpha expression in hypoxic tumor cells

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1 in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1 accumulation in different tumor cell lines. Hypoxia-induced HIF-1 accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F₀F₁-ATPase, prevented hypoxia-induced HIF-1 protein accumulation and had no effect on HIF-1 induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1 content was pronounced in cells exposed to hypoxia (1.5% O₂) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1 regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1 accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function. hypoxia-inducible factor 1; oxygen sensing     

Chlamydia pneumoniae directly interferes with HIF-1alpha stabilization in human host cells

Chlamydiaceae are obligate intracellular bacteria that cause endemic trachoma, sexually transmitted diseases and respiratory infections. The course of the diseases is determined by local inflammatory immune responses and the propensity of the pathogen to replicate within infected host cells. Both features require energy which is inseparably coupled to oxygen availability in the microenvironment. Hypoxia-inducible factor-1 (HIF-1) regulates crucial genes involved in the adaptation to low oxygen concentrations, cell metabolism and the innate immune response. Here we report that Chlamydia pneumoniae directly interferes with host cell HIF-1alpha regulation in a biphasic manner. In hypoxia, C. pneumoniae infection had an additive effect on HIF-1alpha stabilization resulting in enhanced glucose uptake during the early phase of infection. During the late phase of intracellular chlamydial replication, host cell adaptation to hypoxia was actively silenced by pathogen-induced HIF-1alpha degradation. HIF-1alpha was targeted by the chlamydial protease-like activity factor, which was secreted into the cytoplasm of infected cells. Direct interference with HIF-1alpha stabilization was essential for efficient C. pneumoniae replication in hypoxia and highlights a novel strategy of adaptive pathogen-host interaction in chlamydial diseases.     

Quantitation of collagen I,collagen II and aggrecan mRNA and expression of the corresponding proteins in human nucleus pulposus cells in monolayer cultures

Cells that are taken from the nucleus pulposus (NP) and that are allowed to proliferate in monolayer cultures often exhibit changes in their cell morphology and matrix-protein synthesis. However, whether concomitant alterations occur with respect to their mRNA levels for collagen I (CI), collagen II (CII) and aggrecan (AGG) is unclear. In this study, human NP cells from seven individuals were cultured in monolayers and specific mRNAs for CI, CII and AGG were quantified by real-time polymerase chain reaction in fresh NP tissue and during four passages of NP-cell culture. In addition, the presence of CI, CII and AGG protein was determined by immunofluorescence staining of NP cells. We found a significant reduction of CI, CII and AGG mRNA after the initiation of culture in DMEM compared with mRNA levels in fresh NP tissue. During passages 2–4, no further reduction of mRNA levels for CII and AGG was observed. The mRNA level for CI was reduced significantly with duration of culture. Immunofluorescence staining of cultured NP cells revealed expression of CI, CII and AGG protein during the whole culture period. Our data thus demonstrate a reduction of specific mRNA for matrix proteins during the initiation of NP-cell culture but the stable expression of the key matrix proteins, CII and AGG, during further expansion of the cells in monolayers, suggesting no functional changes occur in cultured NP cells. This work was supported by the Medizinisch-wissenschaftlicher Fonds des Buergermeisters der Stadt Wien (grant no. 2177).