Douglas V. Faller

Hypoxia induces endothelin gene expression and secretion in cultured human endothelium.

Hypoxia in vivo is associated with constriction of the distal vasculature in the lung. Uniquely situated at the interface between blood and the vessel wall proper, the vascular endothelium may release vasoactive mediators in the setting of hypoxia. Endothelin-1 is a potent vasoconstrictor released by endothelial cells that could function as a paracrine regulator of vascular tone. We found that physiologic low oxygen tension (PO2 = 30 Torr) increased endothelin secretion from cultured human endothelial cells four to eightfold above the secretion rate at ambient oxygen tension. This increase in secretion was accompanied by a corresponding increase in the transcriptional rate of the preproendothelin gene resulting in increased steady-state mRNA levels of preproendothelin. In contrast, the transcription of a number of other growth-factor-encoding genes, including transforming growth factor-beta, was unaffected by hypoxia. Endothelin transcript production increased within 1 h of hypoxia and persisted for at least 48 h. In addition, the stimulatory effects of low oxygen tension on endothelin mRNA levels were reversible upon reexposure to 21% oxygen environments. These findings suggest a role for endothelin in the control of regional blood flow in the vasculature in response to changes in oxygen tension.

Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia.

The mechanisms by which hypoxia causes vasoconstriction in vivo are not known. Accumulating evidence implicates the endothelium as a key regulator of vascular tone. Hypoxia induces the expression and secretion of endothelin-1 (ET-1), a potent vasoconstrictor in cultured human endothelial cells. We report here that nitric oxide (NO), an endothelial-derived relaxing factor, modifies this induction of ET-1. Whereas low oxygen tension (PO2 = 20-30 Torr) increases ET-1 expression four- to eightfold above that seen at normal oxygen tension (PO2 = 150 Torr), sodium nitroprusside, which releases NO, suppresses this effect. This inhibition of hypoxia-induced ET-1 expression occurs within the first hour of exposure of cells to sodium nitroprusside. Moreover, when the endogenous constitutive levels of NO made by endothelial cells are suppressed using N-omega-nitro-L-arginine, a potent competitive inhibitor of NO synthase, the baseline levels of ET-1 produced in normoxic environments are increased three- to fourfold. The effects of hypoxia and the NO synthase inhibitor on ET-1 expression are additive. The regulation of ET-1 production by NO appears to be at the level of transcription. Similar effects of NO were observed on the expression of the PDGF-B chain gene. PDGF-B expression was suppressed by NO in a hypoxic environment and induced by N-omega-nitro-L-arginine in both normoxic and hypoxic environments. These findings suggest that in addition to its role as a vasodilator, NO may also influence vascular tone via the regulated reciprocal production of ET-1 and PDGF-B in the vasculature.

A short-term trial of butyrate to stimulate fetal-globin-gene expression in the β-globin disorders

BACKGROUND Fetal-globin (gamma-globin) chains inhibit the polymerization of hemoglobin S (sickle hemoglobin) and can functionally substitute for the beta-globin chains that are defective or absent in patients with the beta-thalassemias. Identifying safe mechanisms to stimulate fetal-hemoglobin production is therefore of great interest. Previous studies have shown that administering butyrate selectively stimulates the promoter of the human fetal-globin gene and leads to increases in gamma-globin--gene expression in the developing fetus, cultured cells, and animal models. METHODS To determine whether butyrate can stimulate fetal-globin production in humans, we treated three patients (3 to 13 years old) with sickle cell anemia and three patients (7 to 27 years old) with beta-thalassemia syndromes with a short course of intravenous infusions of arginine butyrate. The drug was infused continuously for either two or three weeks; the initial dose was 500 mg per kilogram of body weight per day. Globin-chain ratios, proportions of reticulocytes producing hemoglobin F (F reticulocytes), and levels of gamma-globin messenger RNA (mRNA) were determined before and during treatment. RESULTS In all six patients, fetal-globin synthesis increased by 6 to 45 percent above pretreatment levels (P < 0.01). The proportion of F reticulocytes increased about twofold, and the level of gamma-globin mRNA increased twofold to sixfold. The increase in gamma-globin synthesis led to improvement in the globin-chain ratios in the patients with thalassemia. The treatment of one patient was extended for seven weeks, and her hemoglobin level increased from 4.7 to 10.2 g per deciliter (2.9 to 6.3 mmol per liter). Side effects were minimal; one patient had a transient increase in serum aminotransferase concentrations. CONCLUSIONS In patients with beta-hemoglobinopathies butyrate, a natural fatty acid, can significantly and rapidly increase fetal-globin production to levels that can ameliorate beta-globin disorders. Further trials of this class of compounds are warranted to determine long-term tolerance and efficacy in patients with sickle cell anemia or beta-thalassemia.

Oxygen tension regulates the expression of the platelet-derived growth factor-B chain gene in human endothelial cells.

Hypoxic states are associated with abnormal proliferation and constriction of the smooth muscle cells surrounding the distal vessels of the lung. In hypoxic as well as in normal states, the endothelial cell layer may play a key role in controlling smooth muscle tone by secreting a number of vasoactive agents. Platelet-derived growth factor (PDGF), produced by endothelial cells, is a major growth factor for vascular smooth muscle cells and a powerful vasoconstrictor. It consists of a disulfide-linked dimer of two related peptides, A and B, that are products of two different genes. We found that hypoxic conditions (0-3% oxygen environments) significantly increased PDGF-B mRNA in cultured human umbilical vein endothelial cells by enhancing the transcriptional rate of this gene. This increase was inversely proportional to oxygen tension and was reversible upon reexposure of cells to a 21% oxygen atmosphere. mRNA levels of PDGF-A were not affected nor was the overall rate of cellular gene transcription increased in response to hypoxia. These studies indicate that endothelial cells are not only capable of sensing oxygen tension, but are also able to discriminate and respond to even small differences in oxygen tension resulting in dramatic upregulation of the PDGF-B chain gene.

ENDOTHELIAL CELL RESPONSES TO HYPOXIC STRESS

1. Changes in the environmental oxygen tension to which cells are exposed in vivo result in physiological and sometimes pathological consequences that are associated with differential expression of specific genes.

Delay in the Fetal Globin Switch in Infants of Diabetic Mothers

In the normal fetus, a switch from production of hemoglobin F (alpha 2 gamma 2) to hemoglobin A (alpha 2 beta 2) occurs at 28 to 34 weeks of gestation. In the fetus with beta-hemoglobinopathy or beta-thalassemia, this switch proceeds despite the morbidity that results when production of beta-globin is abnormal or reduced. Since insulin has recently been shown to induce renewed expression of some inactive genes, we studied globin biosynthesis during the natural evolution of the fetal globin switch under conditions of hyperinsulinemia, which occurs in infants of diabetic mothers. Such infants develop in a hyperglycemic environment, which produces reactive hyperinsulinemia. The normal increase in beta-globin production from pre-switch levels did not occur in 9 of 10 such infants at term, as compared with 11 normal infants, in whom the switch occurred by 36 to 39 weeks of gestation (P less than 0.0001). The delay in the switch from gamma-globin to beta-globin in this unique clinical setting may allow identification of physiologic factors that can modulate developmental gene suppression.

SIRT1 induces EMT by cooperating with EMT transcription factors and enhances prostate cancer cell migration and metastasis

The epithelial-to-mesenchymal transition (EMT) is a crucial program for the invasion and metastasis of epithelial tumors that involves loss of cell–cell adhesion and increased cell mobility; however, mechanisms underlying this transition are not fully elucidated. Here, we propose a novel mechanism through which the nicotinamide adenine dinucleotide-dependent histone deacetylase SIRT1 regulates EMT in prostate cancer cells through cooperation with the EMT inducing transcription factor ZEB1. We found that forced expression of SIRT1 in non-transformed PZ-HPV-7 prostate epithelial cells disrupts the epithelial morphology concomitant with decreased expression of the epithelial marker, E-cadherin, and increased expression of mesenchymal markers. In contrast, silencing SIRT1 in metastatic prostate tumor cells restores cell–cell adhesion and induces a shift toward an epithelial morphology concomitant with increased expression of E-cadherin and decreased expression of mesenchymal markers. We also found that SIRT1 has a physiologically relevant role in endogenous EMT induced by EGF signaling in prostate cancer cells. We propose that the regulation of EMT by SIRT1 involves modulation of, and cooperation with, the EMT inducing transcription factor ZEB1. Specifically, we show that SIRT1 silencing reduces expression of ZEB1 and that SIRT1 is recruited to the E-cadherin proximal promoter by ZEB1 to deacetylate histone H3 and to reduce binding of RNA polymerase II, ultimately suppressing E-cadherin transcription. We thus identify a necessary role for ZEB1 in SIRT1-mediated EMT. Finally, we show that reduction of SIRT1 decreases prostate cancer cell migration in vitro and metastasis in vivo in immunodeficient mice, which is largely independent of any general effects of SIRT1 on prostate cancer growth and survival. We therefore identify SIRT1 as a positive regulator of EMT and metastatic growth of prostate cancer cells and our findings implicate overexpressed SIRT1 as a potential therapeutic target to reverse EMT and to prevent prostate cancer progression.

Phosphorylation of Bcl-2 protein and association with p21Ras in Ras-induced apoptosis.

p21Ras mediates mitogenic responses and also renders cells susceptible to apoptosis after inhibition of protein kinase C (PKC) activity. Ras-induced apoptosis can be blocked by the proto-oncogene bcl-2, but the biochemical or functional nature of Bcl-2 regulation of Ras-induced apoptosis is not understood. We demonstrate that Bcl-2 and p21Ras molecules can be co-immunoprecipitated in Jurkat cells. The level of this association is enhanced when an apoptotic stimulus (inhibition of PKC activity) is delivered. Bcl-2/p21Ras association is coincident with new phosphorylation of the Bcl-2 protein. Inhibition of this phosphorylation prevents protection from apoptosis by Bcl-2, providing a functional correlation to the phosphorylation event. The Bcl-2/p21Ras association cannot be competed by exogenous glutathione S-transferase-Ras fusion protein, suggesting that the endogenous complex may be formed before cell lysis. These results provide a possible mechanism of regulation of Ras-induced apoptosis by Bcl-2.

Short-Chain Fatty Acid Inhibitors of Histone Deacetylases: Promising Anticancer Therapeutics?

Cancer is a disease in which cellular growth regulatory networks are disrupted. Lesions in well-characterized oncogenes and tumor suppressors often contribute to the dysregulation, but recent work has also uncovered the fundamental importance of enzymes that modulate the acetylation status of chromatin to the initiation or progression of cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are known to be involved in physiological cellular processes, such as transcription, cell cycle progression, gene silencing, differentiation, DNA replication, and genotoxic responses, but they are also increasingly being implicated in tumorigenesis. Butyrate is a short-chain fatty acid (SCFA) that acts as a HDAC inhibitor and is being clinically evaluated as an anti-neoplastic therapeutic, primarily because of its ability to impose cell cycle arrest, differentiation, and/or apoptosis in many tumor cell types, and its favorable safety profile in humans. Additionally, HDAC inhibitors could be used in combination with certain established antitumor therapeutics, such as those that target transcription, to augment clinical efficacy, and/or reduce toxicity. The molecular pathways of butyrate and related next-generation synthetic SCFAs in mediating these effects have not been fully elucidated, but HDAC inhibition is associated with regulation of critical cell cycle regulators, such as cyclin D, p21(CIP1/WAF1), and p27(KIP1). It is anticipated that a better understanding of this critical intersection between SCFAs, HDACs, and cell cycle control will lead to the design of novel treatment strategies for neoplasias. This review will summarize some of the recent research in these arenas of HDAC-directed cancer therapy and discuss the potential application of these agents in synergy with current chemotherapeutics.

Prohibitin requires Brg-1 and Brm for the repression of E2F and cell growth

E2F transcription factors play a major role in controlling mammalian cell cycle progression. We recently reported that a potential tumor suppressor, prohibitin, which interacts with retinoblastoma protein (Rb), regulates E2F function and this activity correlates with its growth‐suppressive activity. We show here that prohibitin recruits Brg‐1/Brm to E2F‐responsive promoters, and that this recruitment is required for the repression of E2F‐mediated transcription by prohibitin. Expression of a dominant‐negative Brg‐1 or Brm releases prohibitin‐mediated repression of E2F and relieves prohibitin‐mediated growth suppression. Although prohibitin associates with, and recruits, Brg‐1 and Brm independently of Rb, prohibitin/Brg‐1/Brm‐mediated transcriptional repression requires Rb. A viral oncoprotein, SV40 large T antigen, can reverse prohibitin‐mediated suppression of E2F‐mediated gene transcription, and targets prohibitin through interruption of the association between prohibitin and Brg‐1/Brm without affecting the prohibitin–E2F interaction.