Effects of down-regulated carbonic anhydrase 8 on cell survival and glucose metabolism in human colorectal cancer cell lines.

Carbonic anhydrase 8 (CA8) is a member of the α-carbonic anhydrase family but does not catalyze the reversible hydration of carbon dioxide. In the present study, we examined the effects of CA8 on two human colon cancer cell lines, SW480 and SW620, by suppressing CA8 expression through shRNA knockdown. Our results showed that knockdown of CA8 decreased cell growth and cell mobility in SW620 cells, but not in SW480 cells. In addition, downregulated CA8 resulted in a significant decrease of glucose uptake in both SW480 and SW620 cells. Interestingly, stable downregulation of CA8 decreased phosphofructokinase-1 expression but increased glucose transporter 3 (GLUT3) levels in SW620 cells. However, transient downregulation of CA8 fails to up-regulate GLUT3 expression, indicating that the increased GLUT3 observed in SW620-shCA8 cells is a compensatory effect. In addition, the interaction between CA8 and GLUT3 was evidenced by pull-down and IP assays. On the other hand, we showed that metformin, a first-line drug for type II diabetes patients, significantly inhibited cell migration of SW620 cells, depending on the expressions of CA8 and focal adhesion kinase. Taken together, our data demonstrate that when compared to primary colon cancer SW480 cells, metastatic colon cancer SW620 cells respond differently to downregulated CA8, indicating that CA8 in more aggressive cancer cells may play a more important role in controlling cell survival and metformin response. CA8 may affect glucose metabolism- and cell invasion-related molecules in colon cancer, suggesting that CA8 may be a potential target in future cancer therapy.

Altered glucose metabolism and its association with carbonic anhydrase 8 in Machado-Joseph Disease.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disease. This disorder is caused by polyglutamine (polyQ)-containing mutant ataxin-3, which tends to misfold and aggregate in neuron cells. We previously demonstrated a protective function of carbonic anhydrase 8 (CA8) in MJD disease models and a decreased glycolytic activity associated with down-regulated CA8 in a human osteosarcoma (OS) cell model. Given that a reduction in body weight accompanied by gait and balance instability was observed in MJD patients and transgenic (Tg) mice, in this study, we aimed to examine whether metabolic defects are associated with MJD and whether CA8 expression is involved in metabolic dysfunction in MJD. Our data first showed that glucose uptake ability decreases in cells harboring mutant ataxin-3, but increases in cells overexpressing CA8. In addition, the expressions of glucose transporter 3 (GLUT3) and phosphofructokinase-1 (PFK1) were significantly decreased in the presence of mutant ataxin-3. Consistently, immunohistochemistry (IHC) showed that GLUT3 was less expressed in cerebella of aged MJD Tg mice, indicating that the dysfunction of GLUT3 may be associated with late-stage disease. On the other hand, transient down-regulation of CA8 revealed decreased expressions of GLUT3 and PFK1 in HEK293 cells harboring wild-type (WT) ataxin-3, but no further reduction of GLUT3 and PFK1 expressions were observed in HEK293 cells harboring mutant ataxin-3. Moreover, immunoprecipitation (IP) and immunofluorescence (IF) demonstrated that interactions exist between ataxin-3, CA8 and GLUT3 in MJD cellular and Tg models. These lines of evidence suggest that CA8 plays an important role in glucose metabolism and has different impacts on cells with or without mutant ataxin-3. Interestingly, the decreased relative abundance of Firmicutes/Bacteroidetes (F/B) ratio in the feces of aged MJD Tg mice coincided with weight loss and metabolic dysfunction in MJD. Taken together, our results are the first to demonstrate the effects of CA8 on glucose metabolism and its involvement in the metabolic defects in MJD disease. Further investigations will be required to clarify the underlying mechanisms for the metabolic defects associated with MJD.

Expression and Functional Study of Single Mutations of Carbonic Anhydrase 8 in Neuronal Cells.

Carbonic anhydrase 8 (CA8), an isozyme of α-carbonic anhydrases, lacks the ability to catalyze the reversible hydration of CO2 to bicarbonate and proton. Previous studies have shown that single point mutations of CA8, CA8-S100P, and CA8-G162R, are associated with novel syndromes including congenital ataxia and mild cognitive impairment. Our previous results demonstrated that overexpression of wild type (WT) CA8 promoted cell proliferation, neurite outgrowth, anti-apoptosis, invasion and migration abilities in neuronal cells. In this study, we examined the expressions and functions of CA8-S100P and CA8-G162R in neuroblastoma cells lines, compared with those of WT CA8. Our results show that the protein expressions of mutant CA8-S100P and CA8-G162R were significantly decreased in Neuro-2a and SK-N-SH cells. Interestingly, CA8-S100P demonstrated a significant increase in cell proliferation in both Neuro-2a and SK-N-SH cells. However, both CA8 mutations showed significantly decreased effects on cell protection and migration in SK-N-SH cells. Surprisingly, a significant increase of invasive ability was observed in SK-N-SH cells with overexpression of CA8-S100P as compared with those with overexpression of WT CA8 under retinoic acid (RA) treatment. In addition, we found that Neuro-2a cells with overexpression of CA8-S100P and CA8-G162R showed significantly increased neurite outgrowth. Taken together, our data suggest that the expressions of CA8-S100P and CA8-G162R in neuronal cells alter cell morphology, proliferation, mobility and viability; indicating that the homozygous point mutations of CA8 lead to not only the loss of WT CA8 function, but also the gain of novel functions leading to neuromuscular dysfunction.

Protective roles of carbonic anhydrase 8 in Machado-Joseph Disease.

Machado-Joseph disease (MJD)/Spinocerebellar ataxia type 3 (SCA3) is an inherited neurodegenerative disease that can lead to a regression of motor coordination and muscle control in the extremities. It is known that expansion of CAG repeats encodes abnormally long polyQ in mutant ataxin-3, the disease protein. It is also noted that mutant ataxin-3 interacts with 1,4,5-trisphosphate receptor type 1 (IP3R1) and induces abnormal Ca2+ release. Previously, we have shown a significant increase in the expression of carbonic anhydrase VIII (CA8) in SK-N-SH-MJD78 cells, which are human neuroblastoma cells overexpressing mutant ataxin-3 with 78 glutamine repeats. In the current study, we showed the presence of significantly increased CA8 expression in MJD mouse cerebellum in either early or late disease stage, with a gradual decrease in CA8 expression as the MJD mice naturally aged. By immunofluorescence and immunoprecipitation analysis, we also found that CA8 co-localized and interacted with mutant ataxin-3 in SK-N-SH-MJD78 cells harboring overexpressed CA8 (SK-MJD78-CA8). In addition, we found that SK-MJD78-CA8 cells, as well as cerebellar granule neurons (CGNs) of MJD transgenic (Tg) mouse with overexpressed CA8, were more resistant to reactive oxygen species (ROS) stress than the control cells. Importantly, overexpression of CA8 in SK-MJD78-CA8 cells and in MJD CGNs rescued abnormal Ca2+ release and caused an increase in cell survival. In summary, we demonstrate the protective function of CA8 in MJD disease models and speculate that the declining expression of CA8 following an initial increased expression may be related to the late onset phenomenon of MJD.

Promoter analysis and transcriptional regulation of human carbonic anhydrase VIII gene in a MERRF disease cell model.

Myoclonic epilepsy with ragged-red fibers (MERRF) is a maternally inherited mitochondrial neuromuscular disease. We previously reported a significant decrease of mRNA and protein levels of nuclear DNA-encoded carbonic anhydrase VIII (CA8) in MERRF cybrids harboring A8344G mutation in mitochondrial DNA (mtDNA). In this study, we established a reporter construct of luciferase gene-carrying hCA8 promoter containing several putative transcription factor-binding sites, including GC-box, AP-2 and TATA-binding element in the 5'flanking region of the hCA8 gene. Using a series of mutated hCA8 promoter constructs, we demonstrated that a proximal GC-box, recognized by Sp1 and other Sp family members, may be a key cis-element functioning at the promoter. Additionally, a significant increase of the hCA8 promoter activity was observed in the wild-type and mutant cybrids with over-expression of eGFP-Sp1, but no detectable increase in the CA8 protein expression. In contrast, over-expression of Flag-Sp1 and Flag-Sp4 significantly increased the hCA8 promoter activity as well as endogenous CA8 protein expression in neuron-like HEK-293 T cells. However, down-regulation of Sp1, but not Sp4, in 293 T cells revealed a significant reduction of CA8 expression, suggesting that Sp1 is a predominant transcription factor for regulation of CA8 activity. Furthermore, our data indicate that chromatin structure may be involved in the expression of hCA8 gene in MERRF cybrids. Taken together, these results suggest that Sp1 transactivates hCA8 gene through the proximal GC box element in the promoter region. The key modulator-responsive factor to the mtDNA mutation and how it may affect nuclear hCA8 gene transcription need further investigations.