Defective function of the von Hippel-Lindau (VHL) tumor suppressor ablates proteolytic regulation of hypoxia-inducible factor ␣ subunits (HIF-1␣ and HIF-2␣), leading to constitutive activation of hypoxia pathways in renal cell carcinoma (RCC). Here we report a comparative analysis of the functions of HIF-1␣ and HIF-2␣ in RCC and non-RCC cells. We demonstrate common patterns of HIF-␣ isoform transcriptional selectivity in VHL-defective RCC that show consistent and striking differences from patterns in other cell types. We also show that HIF-␣ isoforms display unexpected suppressive interactions in RCC cells, with enhanced expression of HIF-2␣ suppressing HIF-1␣ and vice-versa. In VHL-defective RCC cells, we demonstrate that the protumorigenic genes encoding cyclin D1, transforming growth factor alpha, and vascular endothelial growth factor respond specifically to HIF-2␣ and that the proapoptotic gene encoding BNip3 responds positively to HIF-1␣ and negatively to HIF-2␣, indicating that HIF-1␣ and HIF-2␣ have contrasting properties in the biology of RCC. In keeping with this, HIF-␣ isoform-specific transcriptional selectivity was matched by differential effects on the growth of RCC as tumor xenografts, with HIF-1␣ retarding and HIF-2␣ enhancing tumor growth. These findings indicate that therapeutic approaches to targeting of the HIF system, at least in this setting, will need to take account of HIF isoform-specific functions.

Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor‐C (VEGF‐C) is a recently described lymphangiogenic factor. Increased expression of VEGF‐C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF‐C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF‐C expression, driven by the rat insulin promoter (Rip), is targeted to β‐cells of the endocrine pancreas. In contrast to wild‐type mice, which lack peri‐insular lymphatics, RipVEGF‐C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic β‐cell tumours that are neither lymphangiogenic nor metastatic. Double‐transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of β‐cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF‐C‐induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases.

Cellular responses to oxygen are increasingly recognized as critical in normal development and physiology, and are implicated in pathological processes. Many of these responses are mediated by the transcription factors HIF-1 and HIF-2. Their regulation occurs through oxygen-dependent proteolysis of the alpha subunits HIF-1alpha and HIF-2alpha, respectively. Both are stabilized in cell lines exposed to hypoxia, and recently HIF-1alpha was reported to be widely expressed in vivo. In contrast, regulation and sites of HIF-2alpha expression in vivo are unknown, although a specific role in endothelium was suggested. We therefore analyzed HIF-2alpha expression in control and hypoxic rats. Although HIF-2alpha was not detectable under baseline conditions, marked hypoxic induction occurred in all organs investigated, including brain, heart, lung, kidney, liver, pancreas, and intestine. Time course and amplitude of induction varied between organs. Immunohistochemistry revealed nuclear accumulation in distinct cell populations of each tissue, which were exclusively non-parenchymal in some organs (kidney, pancreas, and brain), predominantly parenchymal in others (liver and intestine) or equally distributed (myocardium). These data indicate that HIF-2 plays an important role in the transcriptional response to hypoxia in vivo, which is not confined to the vasculature and is complementary to rather than redundant with HIF-1.

Abstract. Oxygen tensions in the kidney are heterogeneous, and their changes presumably play an important role in renal physiologic and pathophysiologic processes. A family of hypoxia-inducible transcription factors (HIF) have been identified as mediators of transcriptional responses to hypoxia, which include the regulation of erythropoietin, metabolic adaptation, vascular tone, and neoangiogenesis. In vitro, the oxygen-regulated subunits HIF-1␣ and -2␣ are expressed in inverse relationship to oxygen tensions in every cell line investigated to date. The characteristics and functional significance of the HIF response in vivo are largely unknown. Highamplification immunohistochemical analyses were used to study the expression of HIF-1␣ and -2␣ in kidneys of rats exposed to systemic hypoxia bleeding anemia, functional anemia (0.1% carbon monoxide), renal ischemia, or cobaltous chloride (which is known to mimic hypoxia). These treatments led to marked nuclear accumulation of HIF-1␣ and -2␣ in different renal cell populations. HIF-1␣ was mainly induced in tubular cells, including proximal segments with exposure to anemia/carbon monoxide, in distal segments with cobaltous chloride treatment, and in connecting tubules and collecting ducts with all stimuli. Staining for HIF-1␣ colocalized with inducible expression of the target genes heme oxygenase-1 and glucose transporter-1. HIF-2␣ was not expressed in tubular cells but was expressed in endothelial cells of a small subset of glomeruli and in peritubular endothelial cells and fibroblasts. The kidney demonstrates a marked potential for upregulation of HIF, but accumulation of HIF-1␣ and HIF-2␣ is selective with respect to cell type, kidney zone, and experimental conditions, with the expression patterns partly matching known oxygen profiles. The expression of HIF-2␣ in peritubular fibroblasts suggests a role in erythropoietin regulation.Sufficient oxygenation is a prerequisite for organ function. However, oxygen delivery to organs and tissue oxygen tensions within organs vary considerably. The kidney is characterized by an interesting paradox with respect to its oxygen supply. Although blood flow is high in relation to organ weight and the arteriovenous oxygen difference is small, shunt diffusion of oxygen and heterogeneous utilization lead to marked oxygen gradients (1,2). Oxygen supply to the renal medulla barely exceeds demand, and medullary oxygen tensions are approximately 10 mmHg (3-6). Cortical oxygen tensions are more heterogeneous but are also frequently less than the venous oxygen tensions (4,6 -8).The effects of oxygen on cellular functions of the kidney are poorly understood. High rates of oxygen consumption in the proximal tubule and thick ascending limb, together with limited oxygen supply, are thought to be responsible for the high sensitivity to ischemic injury (2,9,10). A physiologic function directly related to renal oxygen tensions is the production of erythropoietin (EPO) by peritubular cortical fibroblasts (11-13). Regulation of EPO occurs at the mRN...

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