FIZZ1/RELMα, a Novel Hypoxia-Induced Mitogenic Factor in Lung With Vasoconstrictive and Angiogenic Properties
In a mouse chronic hypoxia model of pulmonary hypertension, we discovered a novel hypoxia-inducible gene in lung, FIZZ1/RELMα, first through a cDNA array analysis and then confirmed by RT-PCR. Western blot and immunohistochemistry revealed that its expression was induced by hypoxia only in lung. The hypoxia-upregulated gene expression was located in the pulmonary vasculature, bronchial epithelial cells, and type II pneumocytes. 3H-thymidine incorporation demonstrated that the recombinant protein stimulated rat pulmonary microvascular smooth muscle cell (RPSM) proliferation dose-dependently ranging from 3.3×10−9 to 3.3×10−8 mol/L. Therefore, we renamed this gene as hypoxia-induced mitogenic factor (HIMF). HIMF strongly activated Akt phosphorylation. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (10 μmol/L) inhibited HIMF-activated Akt phosphorylation. It also inhibited HIMF-stimulated RPSM proliferation. Thus, the PI3K/Akt pathway, at least in part, mediates the proliferative effect of HIMF. Further studies showed that HIMF had angiogenic and vasoconstrictive properties. HIMF increased pulmonary arterial pressure and vascular resistance more potently than either endothelin-1 or angiotensin II.
Pulmonary vascular remodeling, characterized by pulmonary microvascular smooth muscle cell proliferation, is implicated in the development of hypoxic pulmonary arterial hypertension (PAH). To search for the genes that may participate in the pulmonary remodeling, a cDNA microarray analysis (Incyte Genomics; 9415 genes) was performed using lung samples from mice exposed to 10% O2 or room air for 4 days. EST AA712003 was found to be induced by hypoxia.
A literature search revealed that EST AA712003 had just been reported as FIZZ1 (found in inflammatory zone 1), a protein induced in murine lung in an ovalbumin-induced asthma model.1 Besides its induction in the bronchial mucosal epithelial cells, FIZZ1 was also induced in type II pneumocytes and it inhibited NGF-induced survival of DRG neurons and NGF-mediated increase in neuronal CGRP content.1 Holcomb et al also reported that FIZZ1 is a secreted protein sharing the consensus sequence of 10 cysteine residues in the C-terminus (1CX11 2CX8 3CX4 CX3 5CX10 6CX7 CX8 CX9 9C10 C) with two other murine genes expressed respectively in intestinal crypt epithelial (FIZZ2) and white adipose tissue (FIZZ3) and two related human genes (human FIZZ1 and human FIZZ3).1 Later, FIZZ3 was shown to be implicated in type II diabetes mellitus and was renamed as resistin.2 FIZZ1 and FIZZ2 were renamed as resistin-like molecule α (RELMα) and β (RELMβ), respectively.3 Human FIZZ1, however, was renamed as human RELMβ.3 Recently, FIZZ1 was found in macrophages4 and in the stromal vascular fraction of adipose tissue,5 and it inhibited adipocyte differentiation.6 However, the function of FIZZ1 remains unclear.
We hypothesized that FIZZ1 participates in the process of hypoxia-induced pulmonary remodeling. FIZZ1 could be induced at or near the pulmonary vasculature by hypoxia, and the secreted FIZZ1 might have a proliferative effect on the pulmonary vascular smooth muscle cells. We proceeded to validate the microarray result and confirmed that FIZZ1 was markedly induced by hypoxia in the pulmonary vasculature as well as in bronchial epithelial cells and type II pneumocytes. We tested the proliferative role of FIZZ1 in cultured rat pulmonary microvascular smooth muscle cells (RPSM) using a 3H-thymidine incorporation assay with recombinant FIZZ1. FIZZ1 was shown to stimulate RPSM proliferation, and so we renamed FIZZ1 as hypoxia-induced mitogenic factor (HIMF).
Materials and Methods
For a detailed description of animals, hypoxia exposure, cell culture, 3H-thymidine incorporation assays, RT-PCR, recombinant HIMF, Western blots, immunohistochemistry of HIMF, matrigel plug assay, intact chest mouse experiment, rapid amplification of cDNA ends, cloning and analysis of mouse HIMF genomic sequence, and statistical analysis, please see the online data supplement (available at http://www.circresaha.org).
Results and Discussion
HIMF Is Induced in Lung by Hypoxia
RT-PCR results confirmed that hypoxia upregulated HIMF mRNA expression in murine lungs, and the HIMF mRNA induction peaked at 1 day of hypoxia and lasted for 7 days (Figure 1a), corresponding with the period of maximum vascular smooth muscle cell proliferation during development of hypoxic PAH.7
Western blot shows that exposure of mice to hypoxia for 4 days significantly increased HIMF protein expression only in lung (Figure 1b). The immunohistochemistry of mouse lung sections showed that, under normoxic conditions, HIMF protein was not expressed in the pulmonary vasculature, and its expression in epithelial cells was minimal (Figure 1c). Like the ovalbumin-induced asthma model,1 exposure of mice to hypoxia for 4 days increased HIMF protein expression in airway epithelial cells and type II pneumocytes (Figure 1d). However, unlike the ovalbumin-induced asthma model,1 hypoxia markedly increased HIMF expression in the pulmonary vascular cells. HIMF is a secreted protein1 (Figure 2a). Therefore, our results suggest that under hypoxia, HIMF may be secreted from the pulmonary vascular cells as well as the neighboring type II pneumocytes and act on the pulmonary vascular cells through both paracrine and autocrine fashions.
We cloned and sequenced the genomic sequence of mouse HIMF gene (AF516926). Multiple inflammation-related transcription factor binding motifs such as NF-κB, C/EBP, and GAS were found across the genomic sequence, the 5′ and 3′ flanking regions and introns (see online data supplement), suggesting that the expression of HIMF may be regulated by those transcription factors. The mechanism by which hypoxia upregulates the expression of HIMF in lung remains unknown and is worth investigating.
HIMF Stimulates RPSM Proliferation
Chronic hypoxia exposure will result in pulmonary vascular remodeling that is characterized by pulmonary vascular smooth muscle cell migration and proliferation, a major event in the development of hypoxia-induced PAH. We hypothesized that the hypoxia-induced HIMF expression might participate in the pulmonary remodeling. Using a 3H-thymidine incorporation assay, the effect of recombinant HIMF on the proliferation of RPSM was tested. Figure 2b shows that HIMF dose-dependently increased the proliferation of RPSM at concentrations of 3.3×10−9 to 3.3×10−8 mol/L, suggesting that HIMF may play a role in the processes of hypoxia-induced pulmonary vascular remodeling. The exact role of HIMF in the processes of hypoxia-induced pulmonary vascular remodeling needs to be evaluated in vivo.
PI3K/Akt Signal Transduction Pathway at Least in Part Mediates the Proliferative Effect of HIMF
The phosphatidylinositol 3-kinase (PI3K) family of enzymes is activated by a variety of upstream signals and produces 3′ phosphoinositide lipids, which bind to and activate diverse cellular target proteins that ultimately result in the mediation of cellular activities such as proliferation and survival.8 One of the downstream targets of PI3K is the serine/threonine kinase Akt, which mediates cell growth through stabilization of cyclin D1 and downregulation of Cdk inhibitors p27 and p21.8 The PI3K/Akt pathway has been shown to mediate proliferation and migration of human pulmonary vascular smooth muscle cells9 and other vascular smooth muscle cells. To test whether the PI3K/Akt pathway mediates the proliferative effect of HIMF, we first examined whether HIMF could activate the PI3K/Akt pathway. Figure 2c shows that HIMF strongly activated the phosphorylation of Akt. The Akt activation (phosphorylation) peaked at 5 minutes and was sustained through 60 minutes. The PI3K inhibitor LY294002 (10 μmol/L) inhibited HIMF-activated Akt phosphorylation (Figure 2d). Genistein (20 μmol/L) and NF449 (50 μmol/L), which have been shown to inhibit serum-induced RPSM proliferation in the preliminary experiments, however, did not inhibit the Akt activation. LY294002 also inhibited HIMF-stimulated RPSM proliferation (Figure 2e), suggesting that the PI3K/Akt pathway, at least in part, mediates the proliferative effect of HIMF.
HIMF Has Angiogenic and Vasoconstrictive Properties
The PI3K/Akt pathway plays a critical role in the regulation of vascular homeostasis and angiogenesis. HIMF activation of the PI3K/Akt pathway suggests that it may have other effects on the vascular cells besides stimulation of RPSM proliferation. The angiogenic effect of HIMF was evaluated in an in vivo matrigel plug model. HIMF significantly increased the vascular tube formation in the matrigel plug in vivo (Figures 3a through 3d), from 4.3±0.84 to 17.7±1.37/×20 field (n=3, P<0.001), suggesting that it is an angiogenic factor. Whether Akt activation participates in the process of HIMF-induced angiogenesis needs further investigation.
Intravenous injection of HIMF increased the pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR) (Figure 3e). The molar dose-response curves show that HIMF is a more potent pulmonary vasoconstrictor than endothelin-1, angiotensin II, or serotonin (Figure 3e). Injection of FLAG elution buffer had no effect on PAP, suggesting that the vasoconstrictive effect is HIMF specific. Thus, HIMF is a highly potent constrictor of the pulmonary vasculature. The mechanisms underlying the HIMF-induced constriction and the role of HIMF in pulmonary vascular physiology and pathophysiology require further investigation.
In conclusion, HIMF is a hypoxia-induced mitogenic factor in lung with potent angiogenic and pulmonary vasoconstrictive properties. The PI3K/Akt pathway, at least in part, mediates its proliferative effect. These findings warrant thoroughly evaluating the role of HIMF in the development of cardiovascular diseases and further understanding its signal transduction pathways, which may ultimately lead to new avenues for the treatment of cardiovascular diseases.
This work was supported by NIH grants HL 39706 and GM 49111 to R.A.J. We thank Claire F. Levine for assisting in the manuscript preparation and Michael M. Wang for helpful discussions.
This manuscript was sent to Elizabeth G. Nabel, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
↵*Both authors contributed equally to this study.
- Received December 20, 2002.
- Revision received March 7, 2003.
- Accepted April 16, 2003.
Holcomb IN, Kabakoff RC, Chan B, Baker TW, Gurney A, Henzel W, Nelson C, Lowman HB, Wright BD, Skelton NJ, Frantz GD, Tumas DB, Peale FV Jr, Shelton DL, Hebert CC. FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family. EMBO J. 2000; 19: 4046–4055.
Steppan CM, Brown EJ, Wright CM, Bhat S, Banerjee RR, Dai CY, Enders GH, Silberg DG, Wen X, Wu GD, Lazar MA. A family of tissue-specific resistin-like molecules. Proc Natl Acad Sci U S A. 2001; 98: 502–506.
Raes G, De Baetselier P, Noel W, Beschin A, Brombacher F, Hassanzadeh GG. Differential expression of FIZZ1 and Ym1 in alternatively versus classically activated macrophages. J Leukoc Biol. 2002; 71: 597–602.
Blagoev B, Kratchmarova I, Nielsen MM, Fernandez MM, Voldby J, Andersen JS, Kristiansen K, Pandey A, Mann M. Inhibition of adipocyte differentiation by resistin-like molecule α. J Biol Chem. 2002; 277: 42011–42016.
Quinlan TR, Li D, Laubach VE, Shesely EG, Zhou N, Johns RA. eNOS-deficient mice show reduced pulmonary vascular proliferation and remodeling to chronic hypoxia. Am J Physiol Lung Cell Mol Physiol. 2000; 279: L641–L650.
Goncharova EA, Ammit AJ, Irani C, Carroll RG, Eszterhas AJ, Panettieri RA, Krymskaya VP. PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2002; 283: L354–L363.