Induction Of Carbonic Anhydrase Research Articles

Microbially induced calcium carbonate precipitation through CO2 sequestration via an engineered Bacillus subtilis

BackgroundMicrobially induced calcium carbonate precipitation has been extensively researched for geoengineering applications as well as diverse uses within the built environment. Bacteria play a crucial role in producing calcium carbonate minerals, via enzymes including carbonic anhydrase—an enzyme with the capability to hydrolyse CO2, commonly employed in carbon capture systems. This study describes previously uncharacterised carbonic anhydrase enzyme sequences capable of sequestering CO2 and subsequentially generating CaCO3 biominerals and suggests a route to produce carbon negative cementitious materials for the construction industry.ResultsHere, Bacillus subtilis was engineered to recombinantly express previously uncharacterised carbonic anhydrase enzymes from Bacillus megaterium and used as a whole cell catalyst allowing this novel bacterium to sequester CO2 and convert it to calcium carbonate. A significant decrease in CO2 was observed from 3800 PPM to 820 PPM upon induction of carbonic anhydrase and minerals recovered from these experiments were identified as calcite and vaterite using X-ray diffraction. Further experiments mixed the use of this enzyme (as a cell free extract) with Sporosarcina pasteurii to increase mineral production whilst maintaining a comparable level of CO2 sequestration.ConclusionRecombinantly produced carbonic anhydrase successfully sequestered CO2 and converted it into calcium carbonate minerals using an engineered microbial system. Through this approach, a process to manufacture cementitious materials with carbon sequestration ability could be developed.

Carbonic anhydrase integrated into a multimarker approach for the detection of the stress status induced by pollution exposure in Mytilus galloprovincialis: A field case study

The work was addressed to study the sensitivity of the enzyme carbonic anhydrase (CA) to chemical pollution in the hepatopancreas of the bioindicator organism Mytilus galloprovincialis in the context of a multimarker approach in view of ecotoxicological biomonitoring and assessment application. The study was carried out by means of a transplanting experiment in the field, using caged organisms from an initial population exposed in the field in two areas of interest: Augusta-Melilli-Priolo, an heavy polluted industrial site (eastern Sicily, Italy), and Brucoli (eastern Sicily, Italy) an area not affected by any contamination and selected as a reference site.Mussels in Augusta presented a significant increase in the digestive gland CA activity and gene expression compared to the animals caged in the control site of Brucoli. The CA response in animals from the polluted site was paralleled by proliferation/increase in the size of lysosomes, as assessed by Lysosensor green charged cells, induction of metallothionein, up-regulation of hif-α (hypoxia-inducible factor), metabolic changes associated with protein metabolism, and changes in the condition factor. Biological responses data were integrated with information about sediment chemical analysis and metal residue concentration in animal soft tissues.In conclusion, obtained results highlighted the induction of CAs in the hepatopancreas of Mytilus galloprovincialis following to pollution exposure, and demonstrated its suitability to be integrated into a multimarker approach for the detection and characterization of the stress status induced by pollution exposure in this bioindicator organism.

Establishment of hypoxia induction in an in vivo animal replacement model for experimental evaluation of pancreatic cancer.

Transplantation of tumor xenografts to fertilized chicken eggs is a promising animal replacement method, which has successfully been used for xenotransplantation of pancreatic ductal adenocarcinoma (PDA) cells. PDA is characterized by a pronounced tumor hypoxia, which mediates aggressive growth, therapy resistance and cancer stem cell (CSC) features. For in vivo experimental evaluation of hypoxia-targeting therapeutic strategies, the xenografting of tumors to chicken eggs combined with the induction of hypoxia is necessary. However, the chicken embryos do not survive the conventional method of hypoxia induction by a gas mixture of 1% O2, 5% CO2, 94% N2, not even when hypoxia is applied for only 30 min. Therefore, we employed chemical induction of hypoxia by the hypoxia mimetic agent cobalt chloride (CoCl2). Whereas CoCl2 did not further increase tumor growth, it mediated the induction of carbonic anhydrase IX (CAIX) in the tumor xenografts and led to enhanced expression of the human CSC markers CD133, Sox2 and CD44. Side-effects in chicken embryos were not observed as evaluated by H&E staining of embryo-derived liver sections and the determination of the embryo weight. These results suggest the successful induction of hypoxia in chicken eggs and xenografted tumors by CoCl2. For therapeutic intervention and as a control, we treated the eggs with the plant-derived anti-inflammatory agent triptolide, which recently showed promising effects toward hypoxia-induced tumor progression in experimental PDA. Triptolide abolished tumor growth and the CoCl2-induced hypoxic effects, without inducing obvious side-effects. Collectively, our data present a new in vivo animal replacement method for the successful induction of tumor hypoxia in PDA.

Activity enhancement of carbonic anhydrase in Chlamydomonas sp. for effective CO2 sequestration

Present study describes the induction of carbonic anhydrase (CA) activity in Chlamydomonas sp. in presence of calcium carbonate. Results indicated that calcium carbonate increased the activity of extracellular CA enzyme by 14-fold compared to culture grown at 3 % CO2, and by fourfold compared to the culture grown at 0.03 % CO2. CA was purified by gel exclusion followed by ion exchange chromatography. Molecular weight of the purified CA was found to be approximately 28 kDa. The enzyme showed optimum stability in the pH range of 8.0–9.0 and temperature of 35 °C. CA activity was inhibited with Hg2+ and Pb2+, while Zn2+ was found to accelerate its activity. Purified CA was employed for carbonation reaction with CO2 and the calcite produced was characterized by scanning electron microscope and X-ray diffraction. The enhancement in CA activity was found to be reliable, convenient, and may be employed for CO2 sequestration using whole cells or immobilized bioreactor systems.

Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation

Ca-phosphate/hydroxyapatite crystals constitute the mineralic matrix of vertebrate bones, while Ca-carbonate dominates the inorganic matrix of otoliths. In addition, Ca-carbonate has been identified in lower percentage in apatite crystals. By using the human osteogenic SaOS-2 cells it could be shown that after exposure of the cells to Ca-bicarbonate in vitro, at concentrations between 1 and 10 mm, a significant increase of Ca-deposit formation results. The crystallite nodules formed on the surfaces of SaOS-2 cells become denser and larger in the presence of bicarbonate if simultaneously added together with the mineralization activation cocktail (β-glycerophosphate/ascorbic acid/dexamethasone). In parallel, with the increase of Ca-deposit formation, the expression of the carbonic anhydrase-II (CA-II) gene becomes upregulated. This effect, measured on transcriptional level is also substantiated by immunohistological studies. The stimulatory effect of bicarbonate on Ca-deposit formation is prevented if the carbonic anhydrase inhibitor acetazolamide is added to the cultures. Mapping the surface of the Ca-deposit producing SaOS-2 cells by scanning electron microscopy coupled with energy-dispersive X-ray analysis revealed an accumulation of the signals for the element carbon and, as expected, also for phosphorus. Finally, it is shown that ortho-phosphate and hydrolysis products of polyphosphate inhibit CA-II activity, suggesting a feedback regulatory system between the CA-driven Ca-carbonate deposition and a subsequent inactivation of this process by ortho-phosphate. Based on the presented data we suggest that Ca-carbonate deposits act as bioseeds for a downstream Ca-phosphate deposition process. We propose that activators for CA, especially for CA-II, might be beneficial for the treatment of bone deficiency diseases.

Sclerostin Regulates Release of Bone Mineral by Osteocytes by Induction of Carbonic Anhydrase 2

The osteocyte product sclerostin is emerging as an important paracrine regulator of bone mass. It has recently been shown that osteocyte production of receptor activator of NF-κB ligand (RANKL) is important in osteoclastic bone resorption, and we reported that exogenous treatment of osteocytes with sclerostin can increase RANKL-mediated osteoclast activity. There is good evidence that osteocytes can themselves liberate mineral from bone in a process known as osteocytic osteolysis. In the current study, we investigated sclerostin-stimulated mineral dissolution by human primary osteocyte-like cells (hOCy) and mouse MLO-Y4 cells. We found that sclerostin upregulated osteocyte expression of carbonic anhydrase 2 (CA2/Car2), cathepsin K (CTSK/Ctsk), and tartrate-resistant acid phosphatase (ACP5/Acp5). Because acidification of the extracellular matrix is a critical step in the release of mineral from bone, we further examined the regulation by sclerostin of CA2. Sclerostin stimulated CA2 mRNA and protein expression in hOCy and in MLO-Y4 cells. Sclerostin induced a decrease in intracellular pH (pHi) in both cell types as well as a decrease in extracellular pH (pHo) and the release of calcium ions from mineralized substrate. These effects were reversed in the co-presence of the carbonic anhydrase inhibitor, acetozolamide. Car2-siRNA knockdown in MLO-Y4 cells significantly inhibited the ability of sclerostin to both reduce the pHo and release calcium from a mineralized substrate. Knockdown in MLO-Y4 cells of each of the putative sclerostin receptors, Lrp4, Lrp5 and Lrp6, using siRNA, inhibited the sclerostin induction of Car2, Catk and Acp5 mRNA, as well as pHo and calcium release. Consistent with this activity of sclerostin resulting in osteocytic osteolysis, human trabecular bone samples treated ex vivo with recombinant human sclerostin for 7 days exhibited an increased osteocyte lacunar area, an effect that was reversed by the co-addition of acetozolamide. These findings suggest a new role for sclerostin in the regulation of perilacunar mineral by osteocytes.

Differential mRNA expression profiles in proximal tibia of aged rats in response to ovariectomy and low-Ca diet

Both ovariectomized animals and animals fed with Ca-depleted diets are commonly used in vivo models for the investigation of osteoporosis-related bone loss. The present study aimed to study the genomic responses of bone in aged female rats to ovariectomy and dietary Ca deficiency in these models. Aged (11 months old) Sprague–Dawley rats were subjected to bilateral ovariectomy or sham-operation and fed with diets containing different dietary Ca content (LCD, 0.1% Ca or HCD, 1.2% Ca) for 12 weeks. Serum and urine were collected for biochemical marker measurement, and tibias were collected for bone mineral density (BMD) analysis by pQCT as well as for gene expression analysis by real-time PCR. Ovariectomy increased serum N-telopeptides of bone type I collagen (NTx) levels in aged rats fed with HCD ( P < 0.05). In addition, ovariectomy reduced BMD and predicted bone strength of tibial proximal metaphysis in aged rats fed with either LCD or HCD. Dietary Ca deficiency did not alter serum bone-specific alkaline phosphatase (BAP) or NTx levels, but induced a loss of BMD at tibia proximal metaphysis in aged rats. Ovariectomy promoted the mRNA expression of alpha- 1 type I collagen (COL), osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL); and inhibited the mRNA expression of cathepsin K and matrix metalloproteinase-9 (MMP-9) in the proximal tibia of aged rats. Low-Ca diet significantly up-regulated the mRNA expression of COL, core binding factor I (Cbfa1), OPG and carbonic anhydrase II (CAII) in proximal tibia of aged rats. Our study revealed that the genomic responses of bone in proximal tibia to ovariectomy and dietary Ca deficiency were different. The bone loss induced by ovariectomy appears to be mediated primarily by an increase in RANKL mRNA expression; whereas the induction by dietary Ca restriction might be mediated by the induction of carbonic anhydrase II expression.

Induction of carbonic anhydrase IX (CA IX) in rat cardiomyocytes (CM): Link to hypoxia and disruption of oxygen sensing

Induction of carbonic anhydrase IX (CA IX) in rat cardiomyocytes (CM): Link to hypoxia and disruption of oxygen sensing

Induction of carbonic anhydrase IX by hypoxia and chemical disruption of oxygen sensing in rat fibroblasts and cardiomyocytes

CA IX is an active transmembrane carbonic anhydrase isoform functionally implicated in cell adhesion and pH control. Human CA IX is strongly induced by hypoxia and frequently associated with various tumors. In this study, we investigated the expression of the rat CA IX in response to chronic hypoxia and to treatment with chemical compounds that disrupt oxygen sensing, including dimethyloxalylglycine, dimethylester succinate, diazoxide, and tempol. We brought the evidence that expression of CA IX is regulated by hypoxia and hypoxia-mimicking compounds in immortalized Rat2 fibroblasts and BP6 rat fibrosarcoma cells in a cell-type-specific manner. We also demonstrated, for the first time, that CA IX is expressed in hypoxic primary rat cardiomyocytes and in immortalized H9c2 cardiomyocytes exposed to physiological or chemical hypoxia and that CA IX expression is increased in hypoxic rat tissues in vivo. Our findings suggest that CA IX expression is not limited to cancer but may be also induced in other pathological situations associated with ischemia or metabolic disturbances leading to activation of the HIF pathway. These data support the view that rats can represent useful model for studies of CA IX as a component of endogenous protection mechanisms associated with hypoxia or perturbed oxygen sensing.

Inorganic carbon acquisition by Chlamydomonas acidophila across a pH range

Chlamydomonas acidophila Negoro had a higher maximum growth rate upon aeration with 5% CO2 (v/v) than in nonaerated conditions at an external pH above 2. In medium with a pH of 1.0 or 2.0, a decrease in the maximum growth rate was observed upon CO2 aeration in comparison with nonaerated conditions. At both very low and very high external pH conditions, an induction of external carbonic anhydrase was detected; this being more pronounced in CO2-aerated cells than in nonaerated cells. It is therefore suggested that the induction of carbonic anhydrase is part of a stress response in Chlamydomonas acidophila. Comparison of some physiological characteristics of Chlamydomonas acidophila acclimated at pH 2.65 and at pH 6.0, revealed that CO2 aeration increased gross maximum photosynthesis at both pHs, whereas respiration, light acclimation, and photoinhibition were not effected. At pH 2.65, Chlamydomonas acidophila was found to have a carbon-concentrating mechanism under nonaerated conditions, whereas it did not under CO2-aerated conditions at pH 6. The affinity for CO2 use in O2 production was not dependent on CO2 aeration, but it was much lower at pH 6 than it was at pH 2.65. CO2 kinetic characteristics indicate that the photosynthesis of Chlamydomonas acidophila in its natural environment is not limited by inorganic carbon.Key words: Chlamydomonas acidophila, CCM, external carbonic anhydrase, photosynthesis, growth rates, pH stress, CO2.

Expression of membrane-associated carbonic anhydrase isoforms IV, IX, XII, and XIV in the rabbit: induction of CA IV and IX during maturation

Several carbonic anhydrase (CA) isoforms are associated with plasma membranes. It is probable that these enzymes interact with anion transporters to facilitate the movement of HCO3- into or out of the cell. A better knowledge of CA isoform expression in a given tissue would facilitate a systematic examination of any associations with such transporters. We examined the expression of CAs IV, IX, XII, and XIV mRNAs in rabbit tissues, including kidney, heart, lung, skeletal muscle, liver, pancreas, gall bladder, stomach, small intestine, colon, and spleen, using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). CA IV mRNA was mainly in kidney, heart, lung, colon, and gall bladder. CA IX mRNA was restricted to stomach, gall bladder, duodenum, and early jejunum. CA XII mRNA was found in kidney and colon. CA XIV mRNA was localized to heart, lung, skeletal muscle, and liver. The data indicate that there are different patterns of CA expression in various tissues: CA IX was expressed in the proximal gastrointestinal tract, whereas CA XII and CA IV were more distal. CA IV and CA XII are important kidney isoforms. CA XIV was abundant in metabolically active tissues such as liver, heart, lung, and skeletal muscle. Some significant species differences were noted in the expression of some of these isoforms; for example, CA XIV is not expressed in rabbit kidney, despite being abundant in mouse kidney. Maturational studies showed that the expression of CA IX mRNA and protein increased markedly with weaning ( approximately 3-4 postnatal wk) and was well correlated with the maturational expression of the alpha-subunit of the gastric H+,K+-ATPase, suggesting that function of CA IX and the gastric H+ pump might be linked in the digestion of adult foodstuffs. The unique pattern of membrane-bound CA isoforms suggests different functional associations with transporters, depending on the physiological demands on the tissue.

Historical perspective on microalgal and cyanobacterial acclimation to low- and extremely high-CO(2) conditions.

Reports in the 1970s from several laboratories revealed that the affinity of photosynthetic machinery for dissolved inorganic carbon (DIC) was greatly increased when unicellular green microalgae were transferred from high to low-CO(2) conditions. This increase was due to the induction of carbonic anhydrase (CA) and the active transport of CO(2) and/or HCO(3) (-) which increased the internal DIC concentration. The feature is referred to as the 'CO(2)-concentrating mechanism (CCM)'. It was revealed that CA facilitates the supply of DIC from outside to inside the algal cells. It was also found that the active species of DIC absorbed by the algal cells and chloroplasts were CO(2) and/or HCO(3) (-), depending on the species. In the 1990s, gene technology started to throw light on the molecular aspects of CCM and identified the genes involved. The identification of the active HCO(3) (-) transporter, of the molecules functioning for the energization of cyanobacteria and of CAs with different cellular localizations in eukaryotes are examples of such successes. The first X-ray structural analysis of CA in a photosynthetic organism was carried out with a red alga. The results showed that the red alga possessed a homodimeric beta-type of CA composed of two internally repeating structures. An increase in the CO(2) concentration to several percent results in the loss of CCM and any further increase is often disadvantageous to cellular growth. It has recently been found that some microalgae and cyanobacteria can grow rapidly even under CO(2) concentrations higher than 40%. Studies on the mechanism underlying the resistance to extremely high CO(2) concentrations have indicated that only algae that can adopt the state transition in favor of PS I could adapt to and survive under such conditions. It was concluded that extra ATP produced by enhanced PS I cyclic electron flow is used as an energy source of H(+)-transport in extremely high-CO(2) conditions. This same state transition has also been observed when high-CO(2) cells were transferred to low CO(2) conditions, indicating that ATP produced by cyclic electron transfer was necessary to accumulate DIC in low-CO(2) conditions.

Environmentally mediated carbonic anhydrase induction in the gills of euryhaline crustaceans.

The enzyme carbonic anhydrase appears to be a central molecular component in the suite of physiological and biochemical adaptations to low salinity found in euryhaline crustaceans. It is present in high activities in the organs responsible for osmotic and ionic regulation, the gills, and more specifically, the individual gills that are specialized for active ion uptake from dilute sea water. Within those gills carbonic anhydrase is distributed among different subcellular pools, the cytoplasm, mitochondria and microsomes. The cytoplasmic pool represents the largest subcellular fraction of carbonic anhydrase activity, and it is this fraction that undergoes a tenfold induction during acclimation to low salinity. Carbonic anhydrase activity is present in excess of that needed to support the general ion-transport processes, and so it is doubtful that carbonic anhydrase activity itself is a point of short-term regulation in response to salinity changes. Rather, upregulation of carbonic anhydrase appears to be a result of selective gene expression, representing a permanent response to long-term adaptation to low salinity. The exact signal that initiates the induction of carbonic anhydrase, and the pathway through which that signal is transduced to the activation of the carbonic anhydrase gene, are unknown, but two promising avenues of research exist. First, induction of carbonic anhydrase is immediately preceded by hemodilution and subsequent cell swelling, a potential initiating event in the process. Second, recent work indicates that expression of carbonic anhydrase is under the control of a repressor substance, located in the eyestalk, whose effect is removed upon exposure to low salinity.

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO(2).

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO(2) concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1-4 days. In contrast, C. littorale cells which had been grown in 5% CO(2) (5% CO(2)-grown cells) could grow in 40% CO(2) without any lag period. When air-grown cells were transferred to 40% CO(2), the quantum efficiency of PS II (Phi(II)) decreased greatly, while no decrease in Phi(II) was apparent when the 5% CO(2)-grown cells were transferred to 40% CO(2). In contrast to air-grown cells, 5% CO(2)-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO(2)-grown cells to air, photosynthetic susceptibility to 40% CO(2) was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO(2). The intracellular pH value (pH(i)) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO(2) for 1-2 h, but no such decrease in pH(i) was apparent in the presence of EZA. Both air- and 5% CO(2)-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO(2) concentrations, grew in 40% CO(2) without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO(2). No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells.

Carbonic anhydrase is induced prior to arylsulfatase under the simultaneous deprivation of inorganic carbon and sulfate in Chlamydomonas reinhardtii (Volvocales, Chlorophyta)

SUMMARYThe activity of periplasmic arylsulfatase (Ars), which catalyzes the cleavage of sulfate from aromatic sulfur compounds, was detected in cells acclimated to the sulfate‐deficient conditions in a unicellular green alga Chlamydomonas reinhardtii Dangeard, but not in Chlorella, Scenedesmus, Dunaliella and Porphyridium. Upon the transfer of cells to sulfate‐deficient autotrophic media under high‐CO2 conditions, the induction of Ars was observed only in the light, but not in the light with dichlorophenyldimethylurea (DCMU) nor in the dark. However, Ars was induced in the light with DCMU or in the dark when acetate was present as an organic carbon source, but not citrate. Under similar high‐CO2 conditions, high‐CO2 requiring mutants of cia‐3 and cia‐5, whose photosynthetic activities are greatly limited under low CO2, showed much lower level of Ars activities than wild type cells. Under Iow‐CO2 conditions the induction of Ars was greatly suppressed even in wild type and no induction was observed in both mutants. These results suggest that the stimulation of photosynthetic or respiratory carbon metabolism are necessary for the induction of Ars. In contrast, the induction of periplasmic carbonic anhydrase (CA) which was synthesized de novo specifically under CO2‐limited conditions was strongly suppressed by the addition of organic carbon sources, such as acetate and citrate. When cells are subjected to CO2‐limitation and sulfate‐deficiency simultaneously, the induction of CA was initiated immediately, while that of Ars was initiated following the completion of CA induction with an about 4‐h lag. When the concentration of CO2 was suddenly lowered during the induction of Ars, the induction of Ars ceased quickly, and the induction of CA was initiated instead. From these results the induction of CA was suggested to have priority over that of Ars under the dual stress of CO2, and sulfate‐deprivation.

Induction of carbonic anhydrase II expression in osteoclast progenitors requires physical contact with stromal cells.

Carbonic anhydrase II (CA II) expression is vital to normal osteoclast function. We and others have previously reported induction of CA II messenger RNA (mRNA) expression by 1,25(OH)2D3 in myelomonocytic cells and marrow culture. However, since 1,25(OH)2D3 stimulates osteoclast differentiation as well, we wished to separate direct effects of 1,25(OH)2D3 on the CA II gene from the differentiating effects of the hormone. Using primary murine mixed marrow cultures, we measured CA II mRNA expression by RT-PCR. 10 nM 1,25(OH)2D3 dose dependently induced expression of CA II mRNA (4.12 +/- 0.68-fold) at day 4 in culture compared with control with an ED50 of 0.25 nM. When nonadherent marrow cells containing osteoclast progenitors were depleted of stromal cells and exposed to 10 nM 1,25(OH)2D3, CA II mRNA expression was decreased by more than 60%. Coculture of progenitors with ST-2 stromal cells for 3 days with 10 nM 1,25(OH)2D3 stimulated CA II expression by 22 +/- 3.6-fold. 1,25(OH)2D3 stimulated CA II mRNA expression in progenitors separated from ST-2 cells by transwells was insignificant demonstrating that the two cell types must be in physical contact. PTH also stimulated CA II mRNA expression (4.91 +/- 0.01-fold) to a similar degree as seen with 1,25(OH)2D3 treatment. These results demonstrate that induction of CA II in osteoclast progenitors requires their physical communication with stromal cells and is inseparable from the osteoclast differentiation process.

Induction of Carbonic Anhydrase II Expression in Osteoclast Progenitors Requires Physical Contact with Stromal Cells

Carbonic anhydrase II (CA II) expression is vital to normal osteoclast function. We and others have previously reported induction of CA II messenger RNA (mRNA) expression by 1,25(OH)2D3 in myelomonocytic cells and marrow culture. However, since 1,25(OH)2D3 stimulates osteoclast differentiation as well, we wished to separate direct effects of 1,25(OH)2D3 on the CA II gene from the differentiating effects of the hormone. Using primary murine mixed marrow cultures, we measured CA II mRNA expression by RT-PCR. 10 nm 1,25(OH)2D3 dose dependently induced expression of CA II mRNA (4.12 ± 0.68-fold) at day 4 in culture compared with control with an ED50 of 0.25 nm. When nonadherent marrow cells containing osteoclast progenitors were depleted of stromal cells and exposed to 10 nm 1,25(OH)2D3, CA II mRNA expression was decreased by more than 60%. Coculture of progenitors with ST-2 stromal cells for 3 days with 10 nm 1,25(OH)2D3 stimulated CA II expression by 22 ± 3.6-fold. 1,25(OH)2D3 stimulated CA II mRNA expression in progenitors separated from ST-2 cells by transwells was insignificant demonstrating that the two cell types must be in physical contact. PTH also stimulated CA II mRNA expression (4.91 ± 0.01-fold) to a similar degree as seen with 1,25(OH)2D3 treatment. These results demonstrate that induction of CA II in osteoclast progenitors requires their physical communication with stromal cells and is inseparable from the osteoclast differentiation process.

The Regulation of Carbonic Anhydrase and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase by Light and CO2 in Chlamydomonas reinhardtii.

We have investigated the regulation of accumulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activase and the periplasmic carbonic anhydrase (CA) in Chlamydomonas reinhardtii. In algae, the periplasmic CA is required for efficient CO2 fixation when the CO2 concentration is low. These two proteins are affected differently by the CO2 level in the environment. The steady-state level of the ribulose-1,5-bisphosphate carboxylase/oxygenase activase transcript was only slightly and transiently affected by a reduction in ambient CO2 concentration, whereas the CA transcript level was strongly induced by air containing ambient (350 parts per million) CO2 (low CO2) conditions. The transcripts for both proteins showed strong oscillations when the alga was grown under a 12-h light/12-h dark growth regime, with the transcripts encoding these proteins present just before the onset of the light cycle. The observation that the CA transcript was made in the dark was surprising, since earlier reports indicated that active photosynthesis was required for the induction of the periplasmic CA. Further experiments demonstrated that the CA transcript was partially induced under low-CO2 conditions even when the switch to low CO2 was done in the dark. Our results suggest that C. reinhardtii might sense the CO2 concentration in a more direct manner than through C2 or C3 cycle intermediates, which has been previously suggested.

Involvement of photorespiration and glycolate pathway in carbonic anhydrase induction and inorganic carbon concentration in Chlamydomonas reinhardtii

Interaction between induction of carbonic anhydrase (CA) activity, induction of inorganic carbon (Ci) concentrating mechanisms and the photorespiratory glycolate pathway has been studied in wild type 6145c and photorespiratory mutant 18–7F (low in phosphoglycolate phosphatase activity) cells of C. reinhardtii. Cell transfer from high CO2 (5%, v/v) to low CO2 (0.03%) provoked an increase of extracellular and total (extracellular plus intracellular) CA in both wild type and mutant cells. During adaptation to low CO2 conditions, both strains excreted ammonium to the medium at a similar rate in the presence of l‐methionine‐d‐l‐sulfoximine (MSX), an inhibitor of glutamine synthetase (GS). MSX also provoked ammonium excretion by air adapted wild type and mutant cells, even though both strains had high levels of CA activity and of Ci concentrating activities.GS increased in both strains after transfer from high to low CO2 conditions. However, this increase was abolished by aminooxyacetate, an inhibitor of the glyoxylate‐serine aminotransferase, and by glycolaldehyde, an inhibitor of triose phosphate to ribulose 1,5‐bisphosphate conversion. CA synthesis did not occur in the presence of either aminooxyacetate or glycolaldehyde. Algae grown in high CO2 in the presence of aminooxyacetate did not induce Ci concentrating mechanisms. Integration of these three processes, i.e., CA synthesis, Ci‐concentration, and photorespiratory glycolate pathway is proposed in the framework of carbon metabolism of the alga.

Induction of carbonic anhydrase I isozyme precedes the globin synthesis during erythropoiesis in K562 cells

Induction of carbonic anhydrase isozyme I (CA-I) by erythropoietin or hemin was investigated using erythroleukemia (K562) cells. Immunological estimation and purification of carbonic anhydrases showed that untreated K562 cells contained only carbonic anhydrase isozyme II(CA-II), while incubation of the cells with 2 units of erythropoietin (EP) per ml of the incubation medium or with 50 microM hemin resulted in the induction of CA-I. The purified CA-I induced in K562 cells was enzymatically and immunologically identical to that from mature erythrocytes. Flow cytometric analysis showed that incubation of K562 cells with EP as well as hemin induced CA-I at the 3rd h, while alpha-globin was detected at the 8th h. Northern blot analysis of CA-I mRNA using a cloned genomic DNA as a probe showed that mRNA of CA-I was induced by EP. These results suggest that induction of CA-I is regulated at the transcriptional level during developmental changes of erythroid cells, and that CA-I may play a physiologically important role during erythroid differentiation.