Kyle Mansfield

Multiple Factors Affecting Cellular Redox Status and Energy Metabolism Modulate Hypoxia-Inducible Factor Prolyl Hydroxylase Activity In Vivo and In Vitro

ABSTRACT Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-α) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O2)-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O2 deprivation (hypoxia). Using a fusion protein containing the human HIF-1α O2-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1α during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1α accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O2 depletion.

Matrix Regulation of Skeletal Cell Apoptosis ROLE OF CALCIUM AND PHOSPHATE IONS

Previously, we noted that inorganic phosphate (Pi), a major component of bone extracellular matrix, induced osteoblast apoptosis (Meleti, Z., Shapiro, I. M., and Adams, C. S. (2000) Bone (NY) 27, 359–366). Since Ca2+ along with Pi is released from bone during the resorption process, we advanced the hypothesis that Ca2+ modulates Pi-mediated osteoblast apoptosis. To test this hypothesis, osteoblasts were incubated with both ions, and cell death was determined. We noted that a modest increase in the medium Ca2+ concentrations ([Ca2+] e ) of 0.1–1 mm caused a profound and rapid enhancement in Pi-dependent death of cultured osteoblasts. An elevation in [Ca2+] e alone had no effect on osteoblast viability, whereas Ca2+ channel blockers failed to inhibit killing of ion pair-treated cells. These results indicated that Pi-mediated cell death is not dependent on a sustained increase in the cytosolic Ca2+ concentration. Terminal dUTP nick-end labeling analysis and measurement of caspase-3 activity of the ion pair-treated cells suggested that death was apoptotic. Apoptosis was confirmed using caspase-3 and endonuclease inhibitors. The mitochondrial membrane potential and cytosolic Ca2+ status of the treated cells were evaluated. After incubation with [Ca2+ ] e and Pi, a decrease in mitochondrial fluorescence was noted, suggesting that the ions decreased the mitochondrial transmembrane potential. Subsequent to the fall in mitochondrial membrane potential, there was a transient elevation in the cytosolic Ca2+ concentration. Results of the study suggest that the ion pair conspire at the level of the plasma membrane to induce intracellular changes that result in loss of mitochondrial function. The subsequent increase in the cytosolic Ca2+ concentration may trigger downstream events that transduce osteoblast apoptosis.

Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism

In a previous investigation we showed that phosphate ions (Pi) induced apoptosis of terminally differentiated hypertrophic chondrocytes. To explore the mechanism by which Pi induces cell death, we asked the following two questions. First, can we prevent Pi-induced apoptosis by inhibiting plasma membrane Na-Pi cotransport? Second, which specific Na-Pi transporters are expressed in chondrocytes and are they developmentally regulated? Terminally differentiated hypertrophic chondrocytes were isolated from chick tibial cartilage and cell death was measured in the presence of 3-7 mmol/L Pi. To ascertain whether apoptosis was linked to a rise in cellular Pi loading, we examined the effect of phosphonoformic acid (PFA), a competitive inhibitor of Na-Pi cotransport on Pi-induced apoptosis in chondrocytes. We found that 1 mmol/L PFA blocked anion-induced cell death and prevented an increase in the cell Pi content. In a parallel study, we determined that the bisphosphonate, alendronate, also protected chondrocytes from death, albeit at a lower concentration than PFA. Using a DNA end-labeling procedure, we showed that the Pi-treated cells were apoptotic and, as might be predicted, the presence of PFA blocked induction of the death sequence. Next, we examined the expression of two Pi transporters in relation to chondrocyte maturation and anion treatment. We noted that there was expression of the constitutive transporter, Glvr-1, and a type II cotransporter in chick growth plate cells. Although these transport systems are active in terminally differentiated cells, it is probable that the initiation of apoptosis may require the induction of other Pi-transport systems. It is concluded that, at the mineralization front, cell death is linked directly to the elevation in environmental anion concentration and the concomitant rise in intracellular Pi levels.

Induction of Apoptosis in Skeletal Tissues: Phosphate-Mediated Chick Chondrocyte Apoptosis is Calcium Dependent

In an earlier study, we have shown that Pi induced apoptosis of terminally differentiated hypertrophic chondrocytes. To ascertain whether Ca2+ modulates Pi-induced cell death, we asked the following two questions: First, can we prevent Pi-induced apoptosis by removing Ca2+ from the culture medium; alternatively, can we potentiate cell death by increasing the Ca2+ concentration? Second, can we inhibit chondrocyte apoptosis by blocking Pi transport? We also explored the mechanism of apoptosis by evaluating mitochondrial activity and reactive oxygen species (ROS) generation in cells treated with the ion pair. We noted that EDTA and EGTA blocked Pi-induced apoptosis in a dose-dependent manner. While high levels of Ca2+ alone had little effect on chondrocyte viability, the cation enhanced Pi-dependent cell death and greatly increased Pi uptake. When Pi transport was blocked, there was complete inhibition of cell killing. The process of cell death was characterized by mitochondrial hyperpolarization; two hours following apoptogen treatment, there was a significant decrease in the mitochondrial membrane potential. Coincident with the changes in mitochondrial function, there was an increase in intracellular Ca2+ that was maintained throughout the experimental period. A raised Ca2+ signal was observed in blebs at the cell membrane. Finally, we noted that, 75 minutes after treatment with the ion pair, there was a six-fold elevation in ROS levels. This increase declined to baseline values after three hours. Based on these observations, we suggest that, at the cartilage mineralization front, an elevation in local environmental Ca2+ and Pi concentrations modulates oxidative metabolism, and triggers apoptosis of terminally differentiated chondrocytes.

Chondrocyte death is linked to development of a mitochondrial membrane permeability transition in the growth plate.

In the companion article, we reported that the local phosphate (Pi) concentration triggers apoptosis in epiphyseal chondrocytes. The goal of the current investigation was to evaluate the apoptotic process in relationship to the energy status of cells in the growth plate. For these studies, we used sections of the adolescent growth plate, as well as cells isolated from the tissue. We found that there was a maturation‐dependent loss of mitochondrial function in growth plate chondrocytes and these cells generated energy by glycolysis. Since treatment with the uncoupler 2,4‐dinitrophenol as well as the site‐specific inhibitors antimycin A and rotenone failed to elicit a further increase in the activity of the glycolytic pathway, we concluded that oxidative metabolism was minimum in these cells. Flow cytometric studies of growth plate cells and confocal microscopy of growth plate sections using the mitochondrial probes Rh123 and DiOC6(3) provided unequivocal evidence that there was loss of mitochondrial membrane potential in hypertrophic cells. Furthermore, the intrinsic fluorescence of the flavoprotein lipoamide dehydrogenase complex of the electron transport chain revealed that the mitochondria were in an oxidized state. Finally, we assessed Bcl‐2 expression in these cells. Although immunohistochemical and Western blot analysis showed that the chick cells contained a low level of the anti‐apoptotic protein Bcl‐2, reverse transcription‐polymerase chain reaction (RT‐PCR) analysis indicated that transcripts were present in chondrocytes. Based on these observations, we suggest that terminally differentiated chondrocytes undergo a maturation‐dependent loss of mitochondrial function. In concert with the low expression of Bcl‐2, they become sensitive to signals for programmed cell death. We hypothesize that Pi triggers apoptosis in these energy‐compromised cells by promoting a mitochondrial membrane transition, thereby inducing the death process. J. Cell. Physiol. 179:287–296, 1999.

Matrix Regulation of Skeletal Cell Apoptosis II: Role of Arg‐Gly‐Asp‐Containing Peptides

This investigation was based on the assumption that arg‐gly‐asp (RGD)‐containing peptides are released from the extracellular matrix of bone and cartilage during the remodeling cycle. We asked the question: Can RGD peptides influence skeletal cell viability? Primary human osteoblasts, mouse MC‐3T3‐E1 cells, and chick chondrocytes were incubated with purified RGD‐containing peptides and cell viability was determined. The RGD peptide did not kill osteoblasts, chondrocytes, or MC‐3T3‐E1 cells. In contrast, RGDS and GRGDSP peptides killed all three cell types. Osteoblast death was quite rapid, occurring within 6 h of treatment. transferase uridyl mediated nick end labeling (TUNEL) and transmission electron microscopy (TEM) analysis indicated that death was mediated by apoptosis. To learn if mitochondria transduced the death signal, cells were treated with RGDS and organelle function was evaluated using a voltage‐sensitive fluorescent probe. It was observed that there was no net loss of fluorescence and, hence, it was concluded that mitochondria were not the primary effectors of the apoptotic response. Experiments were performed with enzyme inhibitors to determine the import of the caspase pathway on RGDS‐mediated osteoblast apoptosis. Results of these studies, as well as a study conducted using a fluorescent substrate, pointed to caspase 3 mediating the effector stage of the apoptotic process. Finally, using a purified labeled‐RGDS peptide, we showed that the molecule was not restricted by the plasma membrane because it was accumulated in the cytosolic compartment. Results of the investigation support the view that resorption of the extracellular matrix generates peptide products that can induce apoptosis of vicinal cells.

Intrauterine fetal constraint induces chondrocyte apoptosis and premature ossification of the cranial base

Background: The spheno-occipital synchondrosis is an important growth center of the craniofacial skeleton and a primary site of malformation in syndromic forms of craniosynostosis. Clinical and laboratory investigations have demonstrated that premature closure of cranial vault sutures in nonsyndromic craniosynostosis is associated with characteristic alterations in cranial base morphology. However, a causal link between premature fusion of calvarial sutures and changes in the cranial base remains elusive. The purpose of these experiments was to test the hypothesis that intrauterine head constraint produces ultrastructural changes in the spheno-occipital synchondroses of fetal mice. Methods: Fetal constraint was induced through uterine cerclage of six pregnant C57Bl/6 mice on the eighteenth day of gestation. Fetuses were harvested after growing to 24, 48, and 72 hours beyond the normal 20-day gestational period. Between six and nine fetuses were harvested at all time points in both treatment and control groups. The morphology and cell biology of the spheno-occipital synchondroses, in constrained fetuses and unconstrained controls, were examined using hematoxylin and eosin–stained sections. Chondrocyte apoptosis was examined using terminal deoxynucleotidyl transferase-mediated dUDP end-labeling assays and electron microscopy. Results: In nonconstrained animals, the spheno-occipital synchondrosis demonstrated normal architecture and normal chondrocyte morphology at all time points. In contrast, intrauterine constraint resulted in a progressive disruption of the normal cellular architecture of the spheno-occipital synchondrosis over 72 hours, with premature ossification of the synchondrosis. Widespread chondrocyte apoptosis within the synchondrosial growth center was demonstrated by terminal deoxynucleotidyl transferase-mediated dUDP end-labeling assays and electron microscopy. Conclusion: These experiments confirm the ability of intrauterine constraint to induce changes in the morphology and cell biology of the cranial base in synostotic fetuses.

Development of the terminally differentiated state sensitizes epiphyseal chondrocytes to apoptosis through caspase-3 activation

The maturation of epiphyseal chondrocytes is accompanied by dramatic changes in energy metabolism and shifts in proteins concerned with the induction of apoptosis. We evaluated the role of mitochondria in this process by evaluating the membrane potential (Δψm) of chondrocytes of embryonic tibia and the epiphyseal growth plate. We observed that there was a maturation‐dependent change in fluorescence, indicating a fall in the Δψm. The level of mitochondrial Bcl‐2 was decreased during maturation, while in the same time period there was an obvious increase in Bax levels in the mitochondrial fraction of the terminally differentiated chondrocytes. BclxL, another anti‐apoptotic protein, was also robustly expressed in the mitochondrial fraction, but its expression was not dependent on the maturation status of the chondrocytes. We found that caspase‐3 was present throughout the growth plate and in hypertrophic cells in culture. We blocked caspase‐3 activity and found that alkaline phosphatase staining and mineral formation was decreased, and the cells had lost their characteristic shape. Moreover, we noted that the undifferentiated cells were insensitive to elevated concentrations of inorganic phosphate (Pi). It is concluded that during hypertrophy, the change in membrane potential, the increased binding of a pro‐apoptotic protein to mitochondria, and the activation of caspase‐3 serve to prime cells for apoptosis. Only when the terminally differentiated chondrocytes are challenged with low levels of apoptogens there is activation of apoptosis. J. Cell. Physiol. 210: 609–615, 2007.

Maturation‐Dependent Thiol Loss Increases Chondrocyte Susceptibility to Apoptosis

The major aim of the current investigation was to evaluate the role of thiols during chondrocyte maturation and apoptosis. Using a thiol‐sensitive fluorescent probe, we found that in chick growth plate chondrocytes, hypertrophy is accompanied by a decrease in the glutathione content. In this study, we show that the maturation‐dependent loss of thiol, although not causing death of maturing chondrocytes, drastically increases susceptibility to apoptosis by oxidative and nitrosoactive stress. To investigate how the loss of thiol content in cultured chondrocytes affects the expression of the hypertrophic phenotype, we chemically manipulated intracellular thiol levels and analyzed the expression of important maturation markers. We found that thiol depletion causes a decrease in the expression of osteopontin, type X and type II collagen and a significant loss of alkaline phosphatase activity, suggesting that the expression of the hypertrophic phenotype is tightly regulated by redox levels in chondrocytes. Furthermore, severe thiol depletion profoundly affected cell survival under oxidative and nitrosoactive stress. It was concluded that the loss of thiol reserve is not only linked to the expression of the hypertrophic phenotype but also influenced chondrocyte survival, linking chondrocyte maturation and the activation of the apoptotic pathway.