Prolyl-hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype.

Bone is a dynamic environment where cells sense and adapt to changes in nutrient and oxygen availability. Conditions associated with hypoxia in bone are also associated with bone loss. In vitro hypoxia (2% oxygen) alters gene expression in osteoblasts and osteocytes and induces cellular changes including the upregulation of hypoxia inducible factor (HIF) levels. Our studies show that osteoblasts respond to hypoxia (2% oxygen) by enhancing expression of genes associated with adipocyte/lipogenesis phenotype (C/EBPbeta, PPARgamma2, and aP2) and by suppressing expression of genes associated with osteoblast differentiation (alkaline phosphatase, AP). Hypoxia increased HIF protein levels, hypoxic response element (HRE) binding, and HRE-reporter activity. We also demonstrate that prolyl-hydroxylases 2 and 3 (PHD2, PHD3), one of the major factors coordinating HIF degradation under normoxic but not hypoxic conditions, are induced in osteoblasts under hypoxic conditions. To further determine the contribution of PHDs and upregulated HIF activity in modulating osteoblast phenotype, we treated osteoblasts with a PHD inhibitor, dimethyloxaloylglycine (DMOG), and maintained cells under normoxic conditions. Similar to hypoxic conditions, HRE reporter activity was increased and adipogenic gene expression was increased while osteoblastic genes were suppressed. Taken together, our findings indicate a role for PHDs and HIFs in the regulation of osteoblast phenotype.

Gene expression profiling of hypoxia signaling in human hepatocellular carcinoma cells.

Cellular, local, and organismal responses to low O2 availability occur during processes such as anaerobic metabolism and wound healing and pathological conditions such as stroke and cancer. These responses include increases in glycolytic activity, vascularization, breathing, and red blood cell production. These responses are mediated in part by the hypoxia-inducible factors (HIFs), which receive information on O2 levels from a group of iron- and O2-dependent hydroxylases. Hypoxia mimics, such as cobalt chloride, nickel chloride, and deferoxamine, act to simulate hypoxia by altering the iron status of these hydroxylases. To determine whether these mimics are appropriate substitutes for the lower O2 tension evoked naturally, we compared transcriptional responses of a Hep3B cell line using high-density oligonucleotide arrays. A battery of core genes was identified that was shared by all four treatments (hypoxia, cobalt, nickel, and deferoxamine) including glycolytic enzymes, cell cycle regulators, and apoptotic genes. Importantly, cobalt, nickel, and deferoxamine influenced transcription of distinct sets of genes that were not affected by cellular hypoxia. These global responses to hypoxia indicate a balancing act between adaptation and programmed cell death and suggest caution in the use of hypoxia mimics as substitutes for the low O2 tension that occurs in vivo.

The role of hypoxia inducible factor 1alpha in cobalt chloride induced cell death in mouse embryonic fibroblasts.

Cobalt has been widely used in the treatment of anemia and as a hypoxia mimic in cell culture and it is known to activate hypoxic signaling by stabilizing the hypoxia inducible transcription factor 1alpha (HIF1alpha). However, cobalt exposure can lead to tissue and cellular toxicity. These studies were conducted to determine the role of HIF1alpha in mediating cobalt-induced toxicity. Mouse embryonic fibroblasts (MEFs) that were null for the HIF1alpha protein were used to show that HIF1alpha protein plays a major role in mediating cobalt-induced cytotoxicity. Previous work from our lab and others has shown that two BH3 domain containing cell death genes, BNip3 and NIX, are targets of hypoxia signaling. These experiments document that BNip3 and NIX expression is HIF1alpha-dependent, and cobalt induces their expression in a time and dose dependent manner. In addition, their expression is correlated with an increase in BNIP3 and NIX protein. Characteristically, the elevated level of BNIP3 was correlated with an increased presence of chromatin condensation, one marker for cell injury. Interestingly, this increased chromosomal condensation was not coupled to caspase-3 activation as usually seen in a typical apoptotic response. These results show that HIF1alpha is playing a major role in mediating cobalt-induced toxicity in mouse embryonic fibroblasts and may offer a possible mechanism for the underlying pathology of injuries seen in workers exposed to environmental contaminants that can influence the hypoxia signaling system, such as cobalt.

ARA9 modifies agonist signaling through an increase in cytosolic aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of agonists like 2,3, 7,8-tetrachlorodibenzo-p-dioxin. In the current model for AHR signaling, the unliganded receptor is found in the cytosol as part of a complex with a dimer of the 90-kDa heat shock protein and an immunophilin-like molecule, ARA9. In yeast, expression of ARA9 results in an increase in the maximal agonist response and a leftward shift in the AHR dose-response curve. To better understand the mechanism by which ARA9 modifies AHR signal transduction, we performed a series of coexpression experiments in yeast and mammalian cells. Our results demonstrate that ARA9's influence on AHR signaling is not due to inhibition of a membrane pump or modification of the receptor's transactivation properties. Using receptor photoaffinity labeling experiments, we were able to show that ARA9 enhances AHR signal transduction by increasing the available AHR binding sites within the cytosolic compartment of the cell. Our evidence suggests that ARA9's effects are related to its role as a cellular chaperone; i.e. we observed that expression of ARA9 increases the fraction of AHR in the cytosol and also stabilized the receptor under heat stress.

Characterization of the Ah receptor-associated protein, ARA9.

The unliganded aryl hydrocarbon receptor (AHR) is found in a complex with other proteins including the 90-kDa heat shock protein (Hsp90) and a 37-kDa protein we refer to as ARA9. We found that the three tetratricopeptide repeats found in the COOH terminus of ARA9 are necessary and sufficient for interaction with the AHR complex. Conversely, the AHR's "repressor"/Hsp90 binding domain is required for interaction with ARA9. Because ARA9 closely resembles the 52-kDa FK506-binding protein (FKBP52), found in the unliganded glucocorticoid receptor (GR) complex, we compared the binding specificities of ARA9 and FKBP52 for AHR and GR. In co-immunoprecipitation experiments, ARA9 specifically associated with AHR-Hsp90 complex but not with GR-Hsp90 complexes. In addition, ARA9 showed a greater capacity than FKBP52 to associate with AHR-Hsp90 complexes. The biological importance of this interaction was suggested by the observation that in a yeast expression system ARA9 expression enhanced the response of AHR to the agonist beta-napthoflavone, decreasing the EC50 by greater than 5-fold and increasing the maximal response 2.5-fold. In contrast, co-expression of FKBP52 had no effect on AHR signaling. In addition, although ARA9 contains a domain similar to that found in other FK506-binding proteins, ARA9 binding to 3H-FK506 could not be detected. Finally, we have characterized the developmental and expression pattern of ARA9 in the developing mouse embryo and mapped the ARA9 locus to human chromosome 11q13.3.

Regulation of involucrin gene expression by retinoic acid and glucocorticoids.

Involucrin is a major component of the cornified envelope and a marker for terminal differentiation in keratinocytes. We examined hormone regulation of involucrin mRNA expression and transcriptional modulation of the involucrin gene in human keratinocyte cell lines. Dexamethasone enhanced, and retinoic acid decreased, endogenous involucrin mRNA expression in cells treated with these ligands. Functional interactions between each ligand were observed; all-trans or 9-cis retinoic acid reduced dexamethasone enhancement of involucrin expression. Transcriptional control of the involucrin promoter reflected the responses observed for the endogenous gene. All-trans or 9-cis retinoic acid inhibited involucrin expression, and both retinoids antagonized glucocorticoid-mediated induction of reporter gene activity equally well. In contrast, a control promoter was unaffected by hormone treatment, indicating that stabilization of reporter gene mRNA or protein did not account for the observed differences in activity. These results demonstrate that transcriptional control plays a role in regulation of the endogenous involucrin gene by glucocorticoids and retinoids and that DNA sequences within the isolated promoter region confer hormone responsiveness. Hormone-mediated responses mapped to a proximal promoter region that contained a functional AP1 site. Stimulation of AP1 activity by phorbol ester was suppressed by retinoic acid, and cotransfection with a c-jun expression vector reversed the retinoic acid response. Based on these data, we suggest that suppression of involucrin promoter activity by retinoic acid may be mediated through interaction with the AP1 transcriptional complex.

Identification of a functional determinant of differentiation-dependent expression in the involucrin gene.

Involucrin is an integral component of the cornified envelope which is a characteristic feature of the differentiated keratinocyte. Involucrin expression is tightly linked to the onset of differentiation and first expressed in the immediate suprabasal layers of the epidermis. We have identified a transcriptional response element within the distal 5'-flanking region of the involucrin gene which contributes to differentiation-dependent expression. Deletion of this site impairs differentiation-dependent promoter activity in transient transfection analysis, and conversely, this region imparts differentiation-dependent expression to a heterologous promoter. The identified site bears sequence similarity to several AP2-like response elements identified in keratinocyte-specific genes and binds a protein complex (keratinocyte differentiation factor, KDF-1) which is distinct from AP2 by several criteria. The migration of KDF-1 is distinct from AP2 in electrophoretic mobility shift assays, KDF-1 is antigenically unrelated to AP2 since AP2 specific antibodies do not supershift the KDF-1-DNA complex and KDF-1 is poorly competed by oligonucleotides representing consensus AP2 recognition sequences. In addition, the KDF-1 complex is not detected in nuclear extracts derived from human dermal fibroblasts or an enriched population of basal keratinocytes. These findings provide insights to the underlying basis of differentiation-dependent expression of a keratinocyte specific gene.

Myosin heavy chain synthesis is independently regulated in hypertrophy and atrophy of isolated adult cardiac myocytes.

Hypertrophy of isolated adult feline cardiac muscle cells may be induced in culture by either alpha- or beta-adrenergic agonists. However, it has been shown previously that each of these agonists activate different subsets of immediate-early response genes and have different effects on expression of "fetal" protein isoforms and stimulation of protein synthesis. Moreover, in adult feline heart cells, beta-adrenergic agonists, such as isoproterenol, activate sustained synchronous beating and sarcomeric reorganization while alpha-adrenergic agonists, such as phenylephrine, do not. The objective of the present study was to determine whether these differences in proximal signaling events converged in a common signal pathway during activation of contractile protein synthesis. By direct comparisons of actin and myosin heavy chain (HC) synthesis and accumulation following isoproterenol and phenylephrine, it was determined that both agonists stimulate a coordinated accumulation of these proteins during cardiomyocyte growth. However, each agonist stimulated a very different program of contractile protein synthesis. During phenylephrine-induced hypertrophy, actin and myosin HC syntheses were rapidly and coordinately activated and continuously maintained at rates 10-25% greater than untreated cultures. The pattern of myosin HC synthesis following isoproterenol was very much more complex with periods during which it was as much as 40% greater or 25% less than in control cultures. Furthermore, there was no correlation between rates of actin and myosin HC synthesis following isoproterenol. It was concluded that actin and myosin HC syntheses and accumulation were regulated independently and in a very different manner following isoproterenol or phenylephrine. Since protein accumulation was not correlated with synthesis rates during development of hypertrophy, it was also concluded that post-translational mechanisms played a significant role in the maintenance of contractile protein stoichiometry during beta-adrenergic/beating-induced hypertrophy. Myosin HC synthesis also appeared to be independently regulated during cardiomyocyte atrophy induced by the calcium channel blocker nifedipine. Unlike the case in hypertrophy, however, protein balance was not maintained in nifedipine, and the depression of myosin HC synthesis and loss of myosin HC content were much greater than in the case of other contractile proteins.

Regulation of hypertrophy and atrophy in cultured adult heart cells.

Mechanical loading and alpha-adrenergic receptor stimulation have both been shown to induce hypertrophy in isolated neonatal heart cells. The present study examined the effects of adrenergic hormones and contractile activity on the hypertrophic response in isolated adult feline cardiomyocytes maintained for more than 14 days in insulin- and serum-supplemented medium. Measurements of the hypertrophic response included cell size, total protein content, myosin heavy chain content, and the time course of activation of increased protein synthesis. Reactivation of the "fetal" gene program was evaluated by secretion of atrial natriuretic factor (ANF) into the medium. Significant myocyte hypertrophy was induced in both quiescent myocytes treated with alpha 1-adrenergic agonists and in beating myocytes treated with beta-adrenergic agonists. However, there were both quantitative and qualitative differences in the response to each type of stimulation. alpha-Adrenergic agonists promoted an increase in cell size, protein content, and ANF secretion but not myofibrillar reorganization, which was observed only in beating myocytes. In contrast to results reported for neonatal heart cells, determinants of hypertrophy in beating myocytes exceeded those in nonbeating alpha 1-adrenergic agonist-treated heart cells in every parameter examined. In addition, in the case of both beating and alpha-adrenergic stimulation, there were marked time-dependent variations in rates of protein synthesis over the interval of 4 hours to 7 days of treatment with each type of stimulus. Differences were also encountered in correlations between rates of protein synthesis and protein accumulation over this interval. The effect of beating was particularly important both to the reorganization of myofibrillar structure and the metabolism of myosin heavy chain. In cultures in which beating was inhibited with the calcium channel antagonist nifedipine, the loss of myosin heavy chain was significantly greater than that of total protein.

Cultured adult cardiac myocytes maintain protein synthetic capacity of intact adult hearts.

Previous studies have shown that the rates of protein synthesis observed in embryonic and neonatal heart cells in culture are as much as nine times greater than the rates of synthesis observed in the intact adult heart either in situ or in isolated perfusion studies. This study addressed whether adult cardiomyocytes in long-term culture maintain the protein synthetic capacity of the adult myocardium or, rather, whether the protein synthetic capacity expands or falls as adult cardiac myocytes progress in culture. Protein synthesis was evaluated in isolated adult feline cardiomyocytes maintained in serum and insulin-supplemented medium for up to 53 days in vitro. With the use of both pulse- and saturation-labeling techniques it was determined that the rate of protein synthesis in adult cardiomyocytes was maintained at a level very close to that observed in the intact heart for over 1 mo in culture. Saturation-labeling studies indicate a fractional rate of protein synthesis at 6.1%/day and an absolute synthesis rate of 1,300 nmol leucine incorporated.g protein-1.h-1. Pulse-labeling studies revealed an initial increase in protein synthesis rates during adaptation to culture and a further increase after activation of beating and cellular hypertrophy.

Hypertrophy of isolated adult feline heart cells following beta-adrenergic-induced beating.

Catecholamine-induced beating and myocardial hypertrophy were evaluated in isolated adult feline cardiomyocytes maintained in culture for up to 30 days. Adult feline cardiomyocytes were used in this study because they displayed several unique characteristics that facilitated assessment of factors regulating cardiomyocyte hypertrophy in vitro. These characteristics included the following. 1) A single heart provides a high yield of 20-40 x 10(6) calcium-tolerant rod-shaped myocytes. 2) In culture, isolated adult feline cardiomyocytes maintain a stable population of differentiated myocytes that could be maintained without the dramatic loss of cell number, DNA content, or cell structure seen in adult rat cardiomyocyte cultures. 3) Cultured feline cardiomyocytes remained quiescent in culture unless appropriately stimulated to begin beating. 4) Sustained regular beating activity could be readily initiated up to 3 wk in culture by addition of 1 x 10(-5) M isoproterenol, other beta-adrenergic agonists, or agents known to elevate adenosine 3',5'-cyclic monophosphate. Beating could be maintained indefinitely in the presence of isoproterenol, but ceased upon removal of isoproterenol from the medium. Initiation of beating in 7-day-old cultures resulted in a profound restructuring of cardiomyocyte morphology compared with quiescent cultures. Beating heart cells were 66% larger with increased protein content, and they had significantly greater development of striated myofibrillar structure than quiescent myocytes at the same age in culture. We conclude that maintenance of an organized myofibrillar structure in cultured adult cardiac myocytes requires activation of intrinsic beating. Cardiomyocyte hypertrophy also develops following beta-adrenergic activation of beating, but it is unclear whether beating per se is required for inducing hypertrophy in isolated adult cardiomyocytes in vitro.