Vickram Srinivas

Carboxyl-Terminal Transactivation Activity of Hypoxia-Inducible Factor 1α Is Governed by a von Hippel-Lindau Protein-Independent, Hydroxylation-Regulated Association with p300/CBP

ABSTRACT Hypoxia-inducible factor 1 complex (HIF-1) plays a pivotal role in oxygen homeostasis and adaptation to hypoxia. Its function is controlled by both the protein stability and the transactivation activity of its alpha subunit, HIF-1α. Hydroxylation of at least two prolyl residues in the oxygen-dependent degradation domain of HIF-1α regulates its interaction with the von Hippel-Lindau protein (VHL) that targets HIF-1α for ubiquitination and proteasomal degradation. Several prolyl hydroxylases have been found to specifically hydroxylate HIF-1α. In this report, we investigated possible roles of VHL and hydroxylases in the regulation of the transactivation activity of the C-terminal activating domain (CAD) of HIF-1α. We demonstrate that regulation of the transactivation activity of HIF-1α CAD also involves hydroxylase activity but does not require functional VHL. In addition, stimulation of the CAD activity by a hydoxylase inhibitor, hypoxia, and desferrioxamine was severely blocked by the adenoviral oncoprotein E1A but not by an E1A mutant defective in targeting p300/CBP. We further demonstrate that a hydroxylase inhibitor, hypoxia, and desferrioxamine promote the functional and physical interaction between HIF-1α CAD and p300/CBP in vivo. Taken together, our data provide evidence that hypoxia-regulated stabilization and transcriptional stimulation of HIF-1α function are regulated through partially overlapping but distinguishable pathways.

General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia-inducible factor 1alpha.

Avian embryos and neonates acquire passive immunity by transferring maternal immunoglobulins from serum to egg yolk. Despite being a convenient source of antibodies, egg yolk immunoglobulins (IgY) from immunized hens have so far received scant attention in research. Here we report the generation and rapid isolation of IgY from the egg yolk of hens immunized against the α subunit of the human hypoxia‐inducible factor 1 (HIF‐1α). Anti‐HIF‐1α IgY antibodies were affinity purified and tested for their performance in various applications. Abundant HIF‐1α protein was detected by Western blot analysis in nuclear extracts derived from hypoxic cells of human, mouse, monkey, swine, and dog origin whereas in hypoxic quail and frog cells, the HIF‐1α signal was weak or absent, respectively. In electro‐phoretic mobility shift assays, affinity‐purified IgY antibody was shown to recognize the native HIF‐1 (but not the related HIF‐2) complex that specifically binds an oligonucleotide containing the HIF‐1 DNAbinding site. Furthermore, IgY antibody immunoprecipitated HIF‐1α from hypoxic cell extracts. Immunofluorescence experiments using IgY antibody allowed the detection of HIF‐1α in the nucleus of hypoxic COS‐7 cells. For comparison, the application of a mouse monoclonal antibody raised against the identical HIF‐1 α fragment was more restricted. Because chicken housing is inexpensive, egg collection is noninvasive, isolation and affinity purification of IgY antibodies are fast and simple, and the applicability of IgY is widespread, immunization of hens represents an excellent alternative for the generation of polyclonal antibodies. —Camenisch, G., Tini, M., Chilov, D., Kvietikova, I., Srinivas, V., Caro, J., Spielmann, P., Wenger, R. H., Gassmann, M. General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia‐inducible factor 1α. FASEB J. 13, 81–88 (1999)

Regulation of autophagy in human and murine cartilage: Hypoxia-inducible factor 2 suppresses chondrocyte autophagy

OBJECTIVE We have previously demonstrated that the transcription factor hypoxia-inducible factor 1 (HIF-1) promotes the onset of autophagy in chondrocytes. The overall goal of this study was to test the hypothesis that another HIF family transcription factor, HIF-2, modulates the induction of autophagy by chondrocytes. METHODS Expression of HIF-1, HIF-2, and light chain 3 (LC3) in human and murine articular cartilage was visualized by immunohistochemistry. Suppression of HIF-2 was achieved using small interfering RNA technology. Assessments of autophagic flux and lysosomal activity, as well as ultrastructural analysis, were performed in chondrocytes in cell culture. RESULTS HIF-2 was expressed abundantly by cells in human and murine articular cartilage and in the cartilage of mineralizing vertebrae from neonatal mice. Protein levels were reduced in articular cartilage from older mice, in end-plate cartilage from mice, and in chondrocytes from human osteoarthritic (OA) cartilage. HIF-2 was robustly expressed in the prehypertrophic cells of mouse growth cartilage. When HIF-2alpha was silenced, the generation of reactive oxygen species was found to be elevated, with a concomitant decrease in catalase and superoxide dismutase activity. Suppression of HIF-2 was associated with decreased Akt-1 and mammalian target of rapamycin activities, reduced Bcl-xL expression, and a robust autophagic response, even under nutrient-replete conditions. In these silenced chondrocytes, HIF-1 expression was elevated. Decreased HIF-2 expression was associated with autophagy in OA tissues and aging cartilage samples. The autophagic response of chondrocytes in HIF-2alpha-knockout mouse growth plate showed an elevated autophagic response throughout the plate. CONCLUSION Based on these observations, we conclude that HIF-2 is a potent regulator of autophagy in maturing chondrocytes. Our data suggest that this protein acts as a brake on the autophagy-accelerator function of HIF-1.

HIF-1 Regulation of Chondrocyte Apoptosis: Induction of the Autophagic Pathway

The goal of our investigation was to explore the mechanism by which hypoxia regulates growth plate chondrocyte survival. At low O2 tension, chondrocytes were refractory to a staurosporine (i.e., apoptosis-inducing) challenge. To determine whether hypoxic survival was due to the expression of HIF-1, we evaluated the response of HIF silenced cells to staurosporine. Both, silenced cells and control chondrocytes were equally sensitive to the apoptogen challenge. To learn if resistance was mediated by the proteins of the autophagic pathway, we examined the expression of Beclin 1 and LC3. Both proteins were present in the growth plate as well as in N1511 chondrocytes. Moreover, silencing of Beclin 1 resulted in enhanced chondrocyte death. Thus, this gene served to maintain chondrocyte survival activity. Besides serving a cytoprotective role, it is known that autophagy can function in cell death. Accordingly, to ascertain if autophagy might also sensitize cells to apoptosis, we activated autophagy and examined viability following exposure to an apoptogen. Treatment with the autophagy inhibitor 3-methyladenine rendered the chondrocytes refractory to killing, suggesting that sustained autophagy promoted cell death. We next examined expression of BID and caspase-8. When autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID. Finally, we explored the relationship between HIF-1 and Beclin 1. We noted a decrease in Beclin 1 expression and loss of caspase-8 activation in HIF silenced cells and Beclin 1-Bcl-2 association was maintained upon serum starvation. This study indicates that HIF-1 serves to regulate both autophagy and apoptosis.

Autophagy: A New Phase in the Maturation of Growth Plate Chondrocytes Is Regulated by HIF, mTOR and AMP Kinase

The overall goal of the investigation was to examine autophagy in the growth plate and to ascertain how this process was regulated. Herein, we show that in the postmitotic maturing zone of the growth plate, chondrocytes express an autophagic phenotype. This robust and particulate immunohistochemical response provides direct evidence that autophagy is a new and transient stage in the chondrocyte maturation pathway. We found that induction of autophagy was regulated by mTOR, a sensor of cellular metabolism. When mTOR was inhibited, changes in LC3 fluorescence indicated that this kinase regulated development of the autophagic state. To determine if AMP kinase was required for chondrocyte autophagy, we suppressed its expression in N1511 cells using siRNA technology. When these cells were serum starved, a condition that triggers autophagy, there was no change in LC3 distribution. This result confirmed that AMP kinase was required for the induction of the autophagic response. Based on the 2 studies described above, and our previous observation that HIF-1 is required for the induction of autophagy, we put forward the hypothesis that autophagy is regulated by the activities of AMP kinase and mTOR in a HIF-1-dependent manner. Once autophagy is activated, the postmitotic chondrocytes would be expected to remain viable in their unique microenvironment and complete their life cycle.

CHONDROCYTE AUTOPHAGY IS STIMULATED BY HIF-1 DEPENDENT AMPK ACTIVATION AND mTOR SUPPRESSION

The goal of the study is to examine the relationship between the sensor molecules, Hypoxia Inducible Factor-1 (HIF-1), AMP activated Protein Kinase (AMPK) and mammalian Target of Rapamycin (mTOR) in chondrocyte survival and autophagy. We showed that chondrocytes expressed the energy sensor AMPK-1 and that activation increased with maturation. In addition, we showed that thapsigargin treatment activated AMPK and autophagy in a HIF-1-dependent manner. Using serum-starved AMPK-silenced cells, we demonstrated that AMPK was required for the induction of the autophagic response. We also noted a change in chondrocyte sensitivity to apoptogens, due to activation of caspase-8 and cleavage and activation of the pro-apoptotic protein, BID. To test the hypothesis that AMPK signaling directly promoted autophagy, we inhibited AMPK activity in mTOR silenced cells and showed that while mTOR suppression induced autophagy, AMPK inhibition did not block this activity. Based on these findings, it is concluded that because of the micro-environmental changes experienced by the chondrocyte, autophagy is activated by AMPK in a HIF-1-dependent manner.

Hypoxia-inducible factor 1alpha (HIF-1alpha) is a non-heme iron protein. Implications for oxygen sensing.

The hypoxia-inducible factor 1 complex (HIF-1) is involved in the transcriptional activation of several genes, like erythropoietin and vascular endothelial growth factor, that are responsive to the lack of oxygen. The HIF-1 complex is composed of two b-HLH proteins: HIF-1β, that is constitutively expressed, and HIF-1α, that is present only in hypoxic cells. The HIF-1α subunit is continuously synthesized and degraded by the ubiquitin-proteasome under oxic conditions. Hypoxia, transition metals, iron chelators, and several antioxidants stabilize the HIF-1α protein, allowing the formation of the transcriptionally active HIF-1 complex. The mechanisms of oxygen sensing and the pathways leading to HIF-1α stabilization are unclear. Because the involvement of a heme protein oxygen sensor has been postulated, we tested the heme sensor hypothesis by using a luciferase-expressing cell line (B-1), that is highly responsive to hypoxia. Exposure of B-1 cells to carbon monoxide and heme synthesis inhibitors failed to show any effect on the hypoxia responsiveness of these cells, suggesting that heme proteins are not involved in hypoxia sensing. Measurement of iron in recombinantly expressed HIF-1α protein revealed that this protein binds ironin vivo. Iron binding was localized to a 129-amino acid peptide between sequences 529 and 658 of the HIF-1α protein. Although the exact structure of the iron center has not been yet defined, a 2:1 metal/protein molar ratio suggests a di-iron center, probably similar to the one found in hemerythrin. This finding is compatible with a model where redox reaction may occur directly in the iron center of the HIF-1α subunit, affecting its survival inoxic conditions.

Active caspase-3 is required for osteoclast differentiation

Based on our earlier observation that caspase‐3 is present in osteoclasts that are not undergoing apoptosis, we investigated the role of this protein in the differentiation of primary osteoclasts and RAW264.7 cells (Szymczyk KH, et al., 2005, Caspase‐3 activity is necessary for RANKL‐induced osteoclast differentiation. The Proceedings of the 8th ICCBMT). We noted that osteoclast numbers are decreased in long bones of procaspase‐3 knockout mice and that receptor activator of NF‐κB ligand (RANKL) does not promote differentiation of isolated preosteoclasts. In addition, after treatment with inhibitors of caspase‐3 activity, neither the wild‐type primary nor the RAW264.7 cells express TRAP or became multinucleated. We found that immediately following RANKL treatment, procaspase‐3 is cleaved and the activated protein is localized to lipid regions of the plasma membrane and the cytosol. We developed RAW264.7 procaspase‐3 knockdown clonal cell lines using RNAi technology. Again, treatment with RANKL fails to induce TRAP activity or multinucleation. Finally, we evaluated NF‐κB in procaspase‐3 silenced cells. We found that RANKL treatment prevented activation and nuclear translocation of NF‐κB. Together these findings provide direct support for the hypothesis that caspase‐3 activity is required for osteoclast differentiation. J. Cell. Physiol. 209: 836–844, 2006.

AUTOPHAGY IN MINERALIZING TISSUES: MICROENVIRONMENTAL PERSPECTIVES

Chondrocytes in the growth plate and articular cartilage and osteocytes subsumed in Haversian bone exist in environmental niches that are characterized by a limited oxygen supply. In these tissues, cells display a hitherto unrecognized state in which there is evidence of autophagy. The autophagic condition serves to promote cell survival. When the response is triggered, the cell cannibalizes itself to generate energy; if extended, then it can activate Type II apoptosis. We opine that survival is dependent on niche conditions and regulated by crosstalk between mTOR, AMPK and HIF-1 and HIF-2. Recent studies suggest that HIF-2 is a potent regulator of chondrocyte autophagy and that this protein acts as a brake to the stimulatory function of HIF-1. Accordingly, the oxemic state of the tissue, its nutrient supply as well as the energetic state of the cells regulates autophagic flux. From a clinical viewpoint, it may be possible to enhance skeletal cell survival through drugs that modulate the autophagic state and prevent the induction of apoptosis.

Akt-1 mediates survival of chondrocytes from endoplasmic reticulum-induced stress

The unfolded protein response (UPR) is an evolutionary conserved adaptive mechanism that permits cells to react and adjust to conditions of endoplasmic reticulum (ER) stress. In addition to UPR, phosphatidylinositol 3‐kinase (PI3K)/Akt and extracellular signal regulated kinase (ERK) signaling pathways protect a variety of cells from ER stress. The goal of the present study was to assess the susceptibility of chondrocytes to ER stress and to determine the signaling pathways involved in their survival. We found that low concentration of thapsigargin (10 nM) reduced the viability of a chondrocyte cell line (N1511 cells) and that these cells were ∼100 fold more susceptible to thapsigargin‐induced stress than fibroblasts. Interestingly, in thapsigargin and tunicamycin‐stressed chondrocytes induction of the proapoptotic transcription factor CHOP preceded that of the anti‐apoptotic BiP by 12 h. Although both of these agents caused sustained Akt and ERK phosphorylation; inhibition of Akt phosphorylation sensitized chondrocytes to ER stress, while blocking ERK signaling by U0126 had no effect. We found that Akt‐1, but not Akt‐2 or Akt‐3, is predominantly expressed in N1511 chondrocytes. Furthermore, siRNA‐mediated knockdown of Akt‐1 sensitized chondrocytes to ER stress, which was associated with increased capsase‐3 activity and decreased BclXL expression. These data suggest that under condition of ER stress, multiple signaling processes regulate chondrocytes survival‐death decisions. Thus, rapid upregulation of CHOP likely contributes to chondrocyte death, while Akt‐1‐mediated inactivation of caspase 3 and induction of BclXL promotes survival. J. Cell. Physiol. 222: 502–508, 2010.