Although HVEM was originally identified as a cellular receptor mediating the entry of herpes simplex virus (28
), subsequent studies identified its cosignaling functions in mature immune cells (22
). The current study revealed a third biological function of HVEM as a regulator of renal Epo production and erythropoiesis. Induction of HVEM signals by administration of agonistic mAb triggered NO production from renal macrophages and promoted HIF-1α activity in the kidney. Consequently, renal Epo production was elevated, and the expansion and differentiation of erythroid progenitor cells was facilitated. On the other hand, Hvem–/–
mice showed repressed Epo production in response to erythropoietic stress and displayed aggravated anemia. Thus, our findings demonstrated what we believe to be a novel function of HVEM in the regulation of HIF and erythropoiesis.
HIF is the master regulator of adaptive responses to oxygen deprivation, including angiogenesis, erythropoiesis, vasomotor control, energy metabolism, and decisions regulating survival and death (4
). Although HIF activity is regulated by oxygen sensors, such as PHD and FIH, recent advances indicate that oxygen-independent factors also make use of the HIF-mediated gene regulatory system (10
). For instance, inflammatory mediators under infectious conditions, such as LPS and cytokines, upregulate HIF activity regardless of hypoxic stress and promote HIF-driven gene expression, which in turn potentiates antipathogenic effects of innate immune cells (32
). Here, we demonstrated another example of hypoxia-independent HIF regulation mediated by HVEM signaling through NO production by renal macrophages. Although it was reported that enhanced NO expression could stimulate Epo production by decreasing relative blood blow to the kidney (34
), this mechanism was not responsible for the effects observed in our model, as no marked change in renal blood flow was detected in HM3.30-treated mice. In addition, direct measurement of kidney tissue PO2
level also indicated that HVEM-mediated HIF-1α/Epo regulation is a mechanism operating under normoxic conditions.
Our results in experiments attenuating NO activities and depleting macrophages are indicative of an essential role of macrophage-associated NO production in HVEM-mediated erythropoiesis. The stimulatory role of NO in Epo production was previously reported in models using pharmacological methods to increase NO production. For instance, inclusion of S-nitroso-N
-acetyl-DL-penicillamine, an NO donor, to primary rat dorsal root ganglion cultures upregulates Epo transcription in glial cells in a HIF-1α–dependent fashion (35
). In addition, daily injections of sodium nitroprusside, a generator of NO, significantly increased serum Epo levels (36
). Interestingly, our results demonstrated a dispensable role of granulocytes in iNOS expression and erythropoiesis mediated by HVEM signals, in spite of the potential of HVEM signaling to stimulate NO production in granulocytes as well as macrophages (24
). Although a precise mechanism remains to be explored, this result could be explained by distinct thresholds between macrophages and granulocytes in response to HVEM signaling. Indeed, a previous study reported that the HVEM signal strength required for NO production in macrophages is 10 times less than that required in granulocytes (24
). Excepting the enhanced NO production, HM3.30 treatment changed neither the number nor the M1/M2 marker expression pattern of renal macrophages (data not shown). Furthermore, HVEM stimulation did not affect expressions of Epo
, and Pgk1
genes in the liver. Collectively, our present findings suggest that HVEM signaling triggered by HM3.30 selectively facilitates NO production in renal macrophages. Molecular mechanisms of this selectivity need to be explored in future studies.
Our results demonstrated that HVEM stimulation upregulated HIF-1α expression in both renal tubular and peritubular interstitial cells. Although predominant Epo producers under hypoxic conditions are interstitial cells in the kidney (37
), it has also been shown that renal tubular cells have the potential to produce Epo under certain experimental conditions. For instance, ablation of GATA repressor functions triggers Epo expression in renal tubular cells under normoxia (40
). In addition, in renal cell carcinoma patients with the complication of polycythemia, EPO expression is detected in the neoplastic tubular epithelium (41
). At the in vitro culture level, it was also reported that macrophage-derived NO induces HIF-1α expression in coincubated kidney tubular LLC-PK1 cells (42
). Thus, we postulate that HVEM-mediated NO production in renal macrophages induces HIF-1α and Epo expression in adjacent tubular cells as well as interstitial cells under normoxic conditions.
We detected enhanced protein expression of HIF-1α, but not HIF-2α, in the kidneys of HM3.30-treated mice. Although this finding seems inconsistent with previous studies that suggested an important role of HIF-2α in Epo production in response to hypoxic stimuli (43
), it is necessary to consider that the effect of HVEM operates under normoxic conditions. There is compelling evidence that HIF-1α can be upregulated by various stimuli other than hypoxia (12
). In addition, recent studies directly revealed that HIF-1α contributes to NO-mediated Epo production in normoxic condition (35
). Nevertheless, we cannot fully exclude the possibility that weak expression of HIF-2α, which was below the detection limit in our immunoblotting methods, may also contribute to Epo upregulation in our model. Further studies are necessary to address more precise roles of HIF-1α or HIF-2α in HVEM-mediated Epo upregulation.
Although NO-dependent HIF-1α/Epo upregulation plays an essential role in HVEM-mediated erythropoiesis, our results also suggest the possible involvement of other mechanisms. First, ablation of NO activity using iNOS–/–
mice significantly inhibited HVEM-mediated erythropoiesis, but resulted in incomplete attenuation. Thus, NO-independent mechanisms may play some role in the effects of HVEM. Since HVEM is a potent inducer of the NF-κB signaling pathway (20
), it is possible that HVEM upregulates HIF-1α through an NF-κB–dependent mechanism (12
). Second, treatment with anti-Epo neutralizing Ab resulted in incomplete attenuation of erythropoiesis induced by HVEM. Although dose insufficiency of anti-Epo Ab could be a potential cause, it is also possible that HVEM stimulates erythropoiesis through Epo-independent mechanisms. For instance, HVEM may have direct actions on hematopoietic stem cells or myeloid stem cells to promote their potential to differentiate into an erythroid lineage, similar to those observed in Chuvash polycythemia (46
). In addition, a recent study indicated that HIF-1α enhanced the effect of glucocorticoids to promote proliferation of Epo-insensitive erythroid burst-forming unit (BFU-E) progenitors, which also suggests the possibility that HVEM directly affects BFU-E cells to promote erythropoiesis through HIF-1α (47
). In this regard, it should be noted that HVEM was highly expressed on hematopoietic stem cells and progenitor cells (data not shown).
The potential of HVEM signaling to facilitate endogenous Epo production suggests that supplementing HVEM signal can be applied to therapeutic approaches for Epo-responsive diseases, including anemia. Although recombinant EPO and its derivatives have achieved enormous success in treating more than 1,000,000 patients with anemia, these erythropoiesis-stimulating agents (ESAs) still have safety concerns and shortcomings worthy of improvement. It has been revealed that ESA administration increases the risk of thromboembolic events and shortens the survival of cancer patients (48
). Accordingly, the recent guideline summarized by the American Society of Hematology and the American Society of Clinical Oncology recommends that ESAs should be administered at the lowest dose possible and should increase Hb to the lowest concentration possible to avoid transfusions in cancer patients receiving concurrent chemotherapy (52
). Although precise mechanisms of the adverse effects of ESAs have yet to be fully elucidated, nonphysiological pharmacokinetics of exogenously injected recombinant EPO associated with its short half-life may be responsible, at least in part (52
). Administration of agonistic anti-HVEM mAb, on the other hand, induced endogenous Epo upregulation with a relatively long half-life (approximately 48–72 hours; Figure A). Thus, despite the need to proceed with caution, as with other ESAs, stimulation of HVEM signaling may create therapeutic opportunities for Epo-responsive diseases.