Two distinct classes of prechondrogenic cell types in the embryonic limb bud

Differences are demonstrated in the chondrogenic potential of cells derived from the distal and proximal halves of chick wing buds from as early as stage 23, prior to the appearance of overt cartilage differentiation. In high cell density cultures, cells obtained from the distal portions of stage 23 or 24 limb buds are spontaneously chondrogenic in micromass cultures. Cells obtained from the proximal portions, however, become blocked in their differentiation as protodifferentiated cartilage cels, since these cells in micromass cultures make detectable type II collagen, but fail to synthesize significant levels of cartilage proteoglycan or to accumulate an extracellular matrix that will stain for sulfated glycosaminoglycans. Such cultures of proximal limb bud cells can be stimulated to form alcian blue staining nodules by the addition of 1 mM dbcAMP or 50 micrograms/ml ascorbate, or by mixing proximal cells with small numbers of distal cells (1 distal cell to 10 proximal cells). These results demonstrate the existence of two distinct stages among prechondrogenic mesenchyme cells. The earlier stage appears to be able to provide a chondrogenic stimulus to proximal cells.     

Gap junctional communication during limb cartilage differentiation

The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell communication via gap junctions in the regulation of limb cartilage differentiation.     

Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion

Oxidative stress due to excessive reactive oxygen species (ROS) and depleted antioxidants such as glutathione (GSH) can give rise to apoptotic cell death in acutely diabetic hearts and lead to heart disease. At present, the source of these cardiac ROS or the subcellular site of cardiac GSH loss [i.e., cytosolic (cGSH) or mitochondrial (mGSH) GSH] has not been completely elucidated. With the use of rotenone (an inhibitor of the electron transport chain) to decrease the excessive ROS in acute streptozotocin (STZ)-induced diabetic rat heart, the mitochondrial origin of ROS was established. Furthermore, mitochondrial damage, as evidenced by loss of membrane potential, increases in oxidative stress, and reduction in mGSH was associated with increased apoptosis via increases in caspase-9 and -3 activities in acutely diabetic hearts. To validate the role of mGSH in regulating cardiac apoptosis, L-buthionine-sulfoximine (BSO; 10 mmol/kg ip), which blocks GSH synthesis, or diethyl maleate (DEM; 4 mmol/kg ip), which inactivates preformed GSH, was administered in diabetic rats for 4 days after STZ administration. Although both BSO and DEM lowered cGSH, they were ineffective in reducing mGSH or augmenting cardiomyocyte apoptosis. To circumvent the lack of mGSH depletion, BSO and DEM were coadministered in diabetic rats. In this setting, mGSH was undetectable and cardiac apoptosis was further aggravated compared with the untreated diabetic group. In a separate group, GSH supplementation induced a robust amplification of mGSH in diabetic rat hearts and prevented apoptosis. Our data suggest for the first time that mGSH is crucial for modulating the cell suicide program in short-term diabetic rat hearts.     

Depletion of glutathione after gamma irradiation modifies survival

The relationship between the intracellular glutathione (GSH) concentration and the aerobic radiation response was studied in Chinese hamster ovary cells. Various degrees of GSH depletion were produced by exposure to buthionine sulfoximine (BSO) and/or diethyl maleate (DEM). Diethyl maleate did not act as a classical radiosensitizer under the experimental conditions employed, nor did exposure to DEM/BSO nonspecifically affect protein thiols as measured by thiol blotting. Dose-response curves were obtained using cells irradiated in the absence or presence of DEM/BSO, which decreased GSH levels by 90-95%. Exposure to DEM/BSO did not affect the formation of DNA single-strand breaks or DNA-protein crosslinks measured immediately after irradiation performed at ice temperatures. Analysis of survival curves indicated that the Dq was decreased by 18% when GSH depletion occurred prior to, during, and after irradiation. The DEM/BSO exposure did not affect D0. To study postirradiation conditions, cells were exposed to 10 microM DEM prior to and during irradiation, which was performed at ice temperatures. Levels of GSH were depleted by 75% by this protocol. Immediately after irradiation, the cells were rapidly warmed by the addition of 37 degrees C growth medium containing either 10 or 90 microM DEM. Addition of 10 microM DEM after irradiation did not affect the degree of depletion, which remained constant at 75%. In contrast, GSH depletion was increased to 90% 10 min after addition of the 90 microM DEM. Addition of 90 microM DEM after irradiation produced a statistically significant difference in survival compared to addition of 10 microM DEM. In a second depletion protocol, cells were exposed to 100 microM DEM at room temperature for 5 min, irradiated, incubated at 37 degrees C for 1 h, washed, and then incubated in 50 microM BSO for 24 h. This depletion protocol reduced survival by a factor of 2.6 compared to cells not exposed to the combination of DEM/BSO. Survival was not affected if the cells were exposed to the DEM or BSO alone. This was interpreted to indicate that survival was not affected by GSH depletion occurring after irradiation unless depletion was rapid and sustained. The rate of repair of sublethal and potentially lethal damage was measured and found to be independent of the DEM/BSO exposure. These experimental results in addition to previous ones (Freeman and Meredith, Int. J. Radiat. Oncol. Biol. Phys. 13, 1371-1375, 1987) were interpreted to indicate that under aerobic conditions GSH depletion may alter the expression of radiation damage by affecting metabolic fixation.     

The differentiation of normal and muscle-free distal chick limb bud mesenchyme in micromass culture

Distal chick wing bud mesenchyme from stages 19 to 27 embryos has been grown in micromass culture. The behavior of cultures comprising mesenchyme located within 350 microns of the apical ectodermal ridge (distal zone mesenchyme) was compared to that of cultures of the immediately proximal mesenchyme (subdistal zone cultures). In cultures of the distal mesenchyme from stages 21-24 limbs, all of the cells stained immunocytochemically for type II collagen within 3 days, indicating ubiquitous chondrogenic differentiation. At stage 19 and 20, this behavior was only observed in cultures of the distal most 50-100 microns of the limb bud mesenchyme. Between stages 25 and 27, distal zone cultures failed to become entirely chondrogenic. At all stages, subdistal zone cultures always contained substantial areas of nonchondrogenic cells. The different behavior observed between distal zone and corresponding subdistal zone cultures appears to be a consequence of the presence of somite-derived presumptive muscle cells in the latter, since no such difference was observed in analagous cultures prepared from muscle-free wing buds. The high capacity of the distal zone for cartilage differentiation supports a view of pattern formation in which inhibition of cartilage is an important component. However, its consistent behavior in vitro indicates that micromass cultures do not reflect the in vivo differences between the distal zones at different stages. The subdistal region retains a high capacity of cartilage differentiation and the observed behavior in micromass reflects interactions with a different cell population.     

The role of thiols in cellular response to radiation and drugs

Cellular nonprotein thiols (NPSH) consist of glutathione (GSH) and other low molecular weight species such as cysteine, cysteamine, and coenzyme A. GSH is usually less than the total cellular NPSH, and with thiol reactive agents, such as diethyl maleate (DEM), its rate of depletion is in part dependent upon the cellular capacity for its resynthesis. If resynthesis is blocked by buthionine-S,R-sulfoximine(BSO), the NPSH, including GSH, is depleted more rapidly, Cellular thiol depletion by diamide, N-ethylmaleimide, and BSO may render oxygenated cells more sensitive to radiation. These cells may or may not show a reduction in the oxygen enhancement ratio (OER). Human A549 lung carcinoma cells depleted of their NPSH either by prolonged culture or by BSO treatment do not show a reduced OER but do show increased aerobic responses to radiation. Some nitroheterocyclic radiosensitizing drugs also deplete cellular thiols under aerobic conditions. Such reactivity may be the reason that they show anomalous radiation sensitization (i.e., better than predicted on the basis of electron affinity). Other nitrocompounds, such as misonidazole, are activated under hypoxic conditions to radical intermediates. When cellular thiols are depleted peroxide is formed. Under hypoxic conditions thiols are depleted because metabolically reduced intermediates react with GSH instead of oxygen. Thiol depletion, under hypoxic conditions, may be the reason that misonidazole and other nitrocompounds show an extra enhancement ratio with hypoxic cells. Thiol depletion by DEM or BSO alters the radiation response of hypoxic cells to misonidazole. In conclusion, we propose an altered thiol model which includes a mechanism for thiol involvement in the aerobic radiation response of cells. This mechanism involves both thiol-linked hydrogen donation to oxygen radical adducts to produce hydroperoxides followed by a GSH peroxidase-catalyzed reduction of the hydroperoxides to intermediates entering into metabolic pathways to produce the original molecule.     

Cellular glutathione depletion by diethyl maleate or buthionine sulfoximine: no effect of glutathione depletion on the oxygen enhancement ratio

The hypoxic and euoxic radiation response for Chinese hamster lung and A549 human lung carcinoma cells was obtained under conditions where their nonprotein thiols, consisting primarily of glutathione (GSH), were depleted by different mechanisms. The GSH conjugating reagent diethylmaleate (DEM) was compared to DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathionine biosynthesis. Each reagent depleted cellular GSH to less than 5% of control values. A 2-hr exposure to 0.5 mM DEM or a 4- or 24-hr exposure to BSO at 10 or 1 mM, respectively, depleted cellular GSH to less than 5% of control values. Both agents sensitized cells irradiated under air or hypoxic conditions. When GSH levels are lowered to less than 5% by both agents, hypoxic DEM-treated cells exhibited slightly greater X-ray sensitization than hypoxic BSO-treated cells. The D0's for hypoxic survival curves were as follows: control, 4.87 Gy; DEM, 3.22 Gy; and BSO, 4.30 Gy for the V79 cells and 5.00 Gy versus 4.02 Gy for BSO-treated A549 cells. The D0's for aerobic V79 cells were 1.70 Gy versus 1.13 Gy, DEM, and 1.43 Gy for BSO-treated cells. The D0's for the aerobic A549 were 1.70 and 1.20 for BSO-treated cells. The aerobic and anoxic sensitization of the cells results in the OER's of 2.8 and 3.0 for the DEM- and BSO-treated cells compared to 2.9 for the V79 control A549. BSO-treated cells showed an OER of 3.3 versus 3 for the control. Our results suggest that GSH depletion by either BSO or DEM sensitizes aerobic cells to radiation but does not appreciably alter the OER.     

Effect of TGF-beta 1, TGF-beta 2, and bFGF on chick cartilage and muscle cell differentiation

In insulin containing defined medium TGF-beta 1, TGF-beta 2, and bFGF all stimulate chondrogenic differentiation in high-density micromass cultures of distal limb bud mesenchyme cells of chick embryos. In addition bFGF inhibits myogenic differentiation, while TGF-beta 1 and TGF-beta 2 appear to have no effect. TGF-beta 1 and bFGF together act additively to enhance chondrogenesis, while TGF-beta blocks the bFGF inhibitory action on myoblasts, thus allowing them to differentiate. In the absence of insulin, the inhibitory effect of bFGF on muscle cell differentiation is reduced; cartilage differentiation in the presence of the above growth factors is also slightly reduced.     

Direct and progressive differentiation of human embryonic stem cells into the chondrogenic lineage

Treatment of common and debilitating degenerative cartilage diseases particularly osteoarthritis is a clinical challenge because of the limited capacity of the tissue for self‐repair. Because of their unlimited capacity for self‐renewal and ability to differentiate into multiple lineages, human embryonic stem cells (hESCs) are a potentially powerful tool for repair of cartilage defects. The primary objective of the present study was to develop culture systems and conditions that enable hESCs to directly and uniformly differentiate into the chondrogenic lineage without prior embryoid body (EB) formation, since the inherent cellular heterogeneity of EBs hinders obtaining homogeneous populations of chondrogenic cells that can be used for cartilage repair. To this end, we have subjected undifferentiated pluripotent hESCs to the high density micromass culture conditions we have extensively used to direct the differentiation of embryonic limb bud mesenchymal cells into chondrocytes. We report that micromass cultures of pluripotent hESCs undergo direct, rapid, progressive, and substantially uniform chondrogenic differentiation in the presence of BMP2 or a combination of BMP2 and TGF‐β1, signaling molecules that act in concert to regulate chondrogenesis in the developing limb. The gene expression profiles of hESC‐derived cultures harvested at various times during the progression of their differentiation has enabled us to identify cultures comprising cells in different phases of the chondrogenic lineage ranging from cultures just entering the lineage to well differentiated chondrocytes. Thus, we are poised to compare the abilities of hESC‐derived progenitors in different phases of the chondrogenic lineage for cartilage repair. J. Cell. Physiol. 224: 664–671, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of bone-derived chondrogenesis-stimulating activity on embryonic limb mesenchymal cells in vitro

Demineralized bone matrix contains factors which stimulate chondrogenesis and osteogenesis in vivo. A water-soluble extract of bone has been shown to stimulate chondrogenesis in vitro in embryonic limb mesenchymal cells (Syftestad, Lucas & Caplan, 1985). The aim of this study was to analyse the cellular mechanism of the bone-derived chondrogenesis-stimulating activity, with particular attention on how normal requirements for chondrogenesis may be altered. The effects of bovine bone extract (BBE) on chondrogenesis in vitro were studied using micromass cultures of chick limb bud mesenchyme isolated from embryos at Hamburger-Hamilton (HH) stage 23/24, an experimental system which is capable of undergoing chondrogenic differentiation. Bovine diaphyseal long bones were demineralized and extracted with guanidine-HCl to prepare BBE (Syftestad & Caplan, 1984). High-density mesenchyme cultures (30 x 10(6) cells/ml) were exposed to different doses of BBE (0.01-1.0 mg ml-1) and chondrogenesis was quantified based on cartilage nodule number and [35S]sulphate incorporation. BBE was tested on micromass cultures of varying plating densities (2-30 x 10(6) cells/ml), on cultures of 'young' limb bud cells (HH stage 17/18), and on cultures enriched with chondroprogenitor cells obtained from subridge mesoderm. Since poly-L-lysine (PL) has recently been shown (San Antonio & Tuan, 1986) to promote chondrogensis, PL and BBE were introduced together in different doses, in the culture medium, to determine if their actions were synergistic. Our results show that BBE stimulates chondrogenesis in a dose-dependent manner and by a specific, direct action on the chondroprogenitor cells but not in normally non-chondrogenic, low density or 'young' limb bud cell cultures. The effects of PL and BBE are additive and these agents appear to act by separate mechanisms to stimulate chondrogenesis; PL primarily enhances nodule formation, and BBE appears to promote nodule growth.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodismH (bpH) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bpH mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

Diethylmaleate and buthionine sulfoximine, glutathione-depleting agents, differentially inhibit expression of inducible nitric oxide synthase in endotoxemic mice.

Diethylmaleate (DEM) and buthionine sulfoximine (BSO), glutathione (GSH)-depleting agents, reduced the metabolic activity and the protein level of iNOS in both macrophages and hepatocytes activated by lipopolysaccharide (LPS). In this study, we examined the effects of DEM and BSO on iNOS expression in LPS-treated mice under the assumption that the level of GSH may alter the expression of nitric oxide synthase. Serum levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were also determined. DEM markedly decreased the levels of hepatic GSH in response to LPS. Treatment of mice with DEM significantly reduced serum nitrite/nitrate levels and hepatic iNOS protein and mRNA induction by LPS. Although BSO inhibited the level of hepatic GSH in LPS-treated mice, the agent did not alter serum nitrite/nitrate levels and hepatic iNOS expression. DEM completely inhibited an increase of serum IL-1beta level by LPS, whereas BSO failed to inhibit it. Neither DEM nor BSO significantly affected the induction of serum TNF-alpha level by LPS. These results showed that DEM and BSO differentially affect the expression of iNOS in endotoxemic mice, suggesting the possibility that suppression of iNOS expression by DEM may be associated with the inhibition of IL-1beta but not of TNF-alpha.     

Apoptosis vs. necrosis: glutathione-mediated cell death during rewarming of rat hepatocytes

Hypothermia induces injury in its own right, but the mechanisms involved in the cell damage are still unclear. The aim of this study was to test the effects that glutathione (GSH) depletion induces on cell death in isolated rat hepatocytes, kept at 4 degrees C for 20 h, by modulating intracellular GSH concentration with diethylmaleate and buthionine sulfoximine (DEM and BSO). Untreated hepatocytes showed Annexin V stained cells (AnxV(+)), scarce propidium iodide stained cells (PI(+)) and presented a low level of lactate dehydrogenase (LDH) leakage after 20 h at 4 degrees C and rewarming at 37 degrees C. When DEM and BSO were added before cold storage, we observed a few AnXV(+) cells and an increase in PI(+) cells associated with LDH release in the incubation medium. Conversely, the addition of DEM and BSO only during rewarming caused a marked increase in cell death by apoptosis. Production of reactive oxygen species (ROS) and thiobarbituric acid species (TBARS), associated with a decrease in GSH concentrations, was higher when DEM and BSO were added before cold storage. Cells treated with DEM and BSO before cold storage showed lower ATP energy stores than hepatocytes treated with DEM and BSO only during rewarming. Pretreatment of hepatocytes with deferoxamine protected against apoptotic and necrotic morphology in conditions of GSH depletion. These results suggest that pretreatment of hepatocytes with DEM and BSO before cold storage induces necrosis, while the treatment of hepatocytes only during rewarming increases apoptosis. In both conditions, iron represents a crucial mediator of cell death.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Abstract. Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodism^H ( bp ^H ) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bp ^H mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-727) cells to the cytotoxic effects of Cd⁺⁺. The effects of GSH depletion on Cd⁺⁺ accumulation and Cd⁺-induced metallothionein (MT) content were investigated to determine the possible role of these Cd⁺⁺ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd⁺⁺-induced accumulation of exogenous³⁵s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd⁺⁺ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd⁺⁺ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd⁺⁺, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd⁺⁺-induced MT accumulation rates.Abbreviations GSH glutathione - MT metallothionein - BSO DL-buthionine-[S,R]-sulfoximine - DMSO dimethyl sulfoximine - DEM diethyl maleate - NP-40 nonidet-P40 - PBS phosphate buffered saline - HBSS Hank's balanced salt solution - DTT dithiothreitol 3. This work was presented in part at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1–5, 1988.     

Involvement of Frzb-1 in mesenchymal condensation and cartilage differentiation in the chick limb bud

In developing limb bud, mesenchymal cells form cellular aggregates called "mesenchymal condensations". These condensations show the prepattern of skeletal elements of the limb prior to cartilage differentiation. Roles of various signaling molecules in chondrogenesis in the limb bud have been reported. One group of signaling factors includes the Wnt proteins, which have been shown to have an inhibitory effect on chondrogenesis in the limb bud. Therefore, regulation of Wnt activity may be important in regulating cartilage differentiation. Here we show that Frzb-1, which encodes a secreted frizzled-related protein that can bind to Wnt proteins and can antagonize the activity of some Wnts, is expressed in the developing limb bud. At early stages of limb development, Frzb-1 is expressed in the ventral core mesenchyme of the limb bud, and later Frzb-1 expression becomes restricted to the central core region where mesenchymal condensations occur. At these stages, a chondrogenic marker gene, aggrecan, is not yet expressed. As limb development proceeds, expression of Frzb-1 is detected in cartilage primordial cells, although ultimately Frzb-1 expression is down-regulated. Similar results were obtained in the recombinant limb bud, which was constructed from dissociated and re-aggregated mesenchymal cells and an ectodermal jacket with the apical ectodermal ridge. In addition, Frzb-1 expression preceded aggrecan expression in micromass cultures. These results suggest that Frzb-1 has a role in condensation formation and cartilage differentiation by regulating Wnt activity in the limb bud.     

The role of glutathione in lymphocyte activation. I. Comparison of inhibitory effects of buthionine sulfoximine and 2-cyclohexene-1-one by nuclear size transformation

The role of glutathione (GSH) in lectin-induced lymphocyte activation can be studied by quantitating lectin-induced nuclear size transformation in the presence of variable degrees of GSH depletion. Buthionine sulfoximine (BSO) inhibits intracellular GSH synthesis by inhibition of the enzyme gamma-glutamyl-cysteine synthetase. By combining endogenous GSH depletion in cell cultures with BSO-induced inhibition of GSH synthesis, lectin-induced lymphocyte activation can be studied at various concentrations of soluble intracellular GSH. With this approach, the percentage of lymphocytes undergoing a nuclear size transformation is minimally affected despite depletion of soluble intracellular GSH to 0.27 nmol/10(7) cells (PBL), which represents approximately 95% depletion of intracellular GSH. When soluble intracellular GSH is depleted to undetectable levels (less than 0.10 nmol/10(7) cells) there is a 10 to 12% reduction in the number of cell nuclei transformed. However, in all BSO-pretreated cultures the lectin-induced nuclear size transformation is intermediate between resting and blast-transformed lymphocytes, suggesting only partial (or aborted) activation. The partial activation response observed in BSO-pretreated cultures may be due to mobilization of the protein-bound pool of GSH, which is relatively resistant to depletion by BSO. That the inhibition of full blast transformation is truly due to GSH depletion was proven by experiments in which GSH was repleted exogenously and a full blast transformation was restored. The results of previous work in our laboratory had shown that the sulfhydryl-reactive agent 2-cyclohexene-1-one (2-CHX) was a potent inhibitor of activation at soluble intracellular GSH concentrations well above 0.27 nmol/10(7) PBL. In the present study, the dose-dependent inhibition of activation by 2-CHX was confirmed, but it was shown that the degree of inhibition caused by 2-CHX could be at least partially dissociated from the level of intracellular GSH present at the time of lectin addition and that the inhibitory potential of 2-CHX exceeded that of BSO at comparable levels of soluble intracellular GSH. Thus, the inhibitory properties of 2-CHX cannot be accounted for solely on the basis of GSH depletion.     

Lack of chondrogenic expression in mouse limb bud micromass cultures exposed to exogenous beta galactosidase or N-acetyl-beta-glucosaminidase

The effect of two exoglycosidases, beta-galactosidase and N-acetyl-beta-glucosaminidase (GlcNAc-ase) on chondrogenic expression of stage 19 mouse limb bud micromass cultures was investigated. Chondrogenic expression was monitored by Alcian blue staining and immunofluorescent localization of cartilage-specific proteoglycan and type II collagen. Chondrogenesis was inhibited by exposure to 0.1 U/ml beta-galactosidase or 0.025 U/ml GlcNAc-ase for 24 h or longer in culture. The effect of both enzymes was concentration and time dependent. Exoglycosidic hydrolysis of galactose or N-acetylglucosamine was substantiated by treatment with HRP-conjugated peanut agglutinin and succinylated wheat germ agglutinin, respectively. Cells treated with beta-galactosidase appeared to be flattened with a stellate morphology, whereas GlcNAc-ase-treated cells were bipolar forming ridge-like mounds that had a directional orientation. The antichondrogenic effect was not alleviated when the cells were induced to assume a spherical shape upon treatment with cytochalasin D. DNA measurements indicated that the lack of chondrogenic expression was not related to cell attachment or cell proliferation. These data support the hypothesis that the expression of specific terminal sugars on cell surface glycoconjugates of limb bud cells represents an important component of the chondrogenic process.     

Conditional Ablation of the Heparan Sulfate-synthesizing Enzyme Ext1 Leads to Dysregulation of Bone Morphogenic Protein Signaling and Severe Skeletal Defects

Increasing evidence indicates that heparan sulfate (HS) is an integral component of many morphogen signaling pathways. However, its mechanisms of action appear to be diverse, depending on the type of morphogen and the developmental contexts. To define the function of HS in skeletal development, we conditionally ablated Ext1, which encodes an essential glycosyltransferase for HS synthesis, in limb bud mesenchyme using the Prx1-Cre transgene. These conditional Ext1 mutant mice display severe limb skeletal defects, including shortened and malformed limb bones, oligodactyly, and fusion of joints. In developing limb buds of mutant mice, chondrogenic differentiation of mesenchymal condensations is delayed and impaired, whereas the area of differentiation is diffusely expanded. Correspondingly, the distribution of both bone morphogenic protein (BMP) signaling domains and BMP2 immunoreactivity in the mutant limb mesenchyme is broadened and diffuse. In micromass cultures, chondrogenic differentiation of mutant chondrocytes is delayed, and the responsiveness to exogenous BMPs is attenuated. Moreover, the segregation of the pSmad1/5/8-expressing chondrocytes and fibronectin-expressing perichondrium-like cells surrounding chondrocyte nodules is disrupted in mutant micromass cultures. Together, our results show that HS is essential for patterning of limb skeletal elements and that BMP signaling is one of the major targets for the regulatory role of HS in this developmental context.