Role of p53 in lung tissue remodeling

Abstract

The tumor suppressor gene p53 regulates a wide range of cellular processes including cell cycle progression, proliferation, apoptosis and tissue development and remodeling. Lung cell apoptosis and tissue remodeling have critical roles in many lung diseases. Abnormal proliferation or resistance to apoptosis of lung cells will lead to structural changes of many lung tissues, including the pulmonary vascular wall, small airways and lung parenchyma. Among the many lung diseases caused by vascular cell apoptosis and tissue remodeling are chronic obstructive pulmonary disease, bronchial asthma and pulmonary arterial hypertension. Recent advances in biology and medicine have provided new insights and have resulted in new therapeutic strategies for tissue remodeling in human and animal models. This review is focused on lung disease susceptibility associated with the p53 pathway and describes molecular mechanisms upstream and downstream of p53 in lung tissue remodeling. Improved understanding of structural changes associated with pulmonary vascular remodeling and lung cell apoptosis induced by the p53 pathway may new provide therapeutic targets.

Key Words: Chronic obstructive pulmonary disease, Pulmonary hypertension, Asthma, p21, Mouse double minute 2 homolog

Core tip: The activated p53 protein and its associated pathway play a pivotal role in tissue remodeling in chronic obstructive pulmonary disease, asthma and pulmonary hypertension. p53 protein regulates numerous genes and proteins associated with cell cycle arrest and apoptosis. In response to oxidative stress or hypoxia, p53 can become stabilized and activate signal transduction towards lung tissue remodeling and functional loss.

INTRODUCTION

The p53 tumor suppressor protein regulates a number of cellular functions such as cell cycle arrest, gene transcription, and apoptosis in response to DNA damage[1,2]. p53 was first identified in 1979 as an oncogene[3], and later study revealed that the gene is a tumor suppressor in nature[4]. p53 is a regulator of transcription which will activate the target gene transcription. Generally, p53 binds as a tetramer to the promoter regions of target genes, and the most known about p53 target genes including p21, a cyclin-dependent kinase inhibitor, mouse double minute 2 homolog (MDM2), p53 up-regulated modulator of apoptosis (Puma), and Bcl-2-associated X protein (Bax), which are known as apoptosis inducers[5].

In normal cells, the p53 expression is at very low levels, which is controlled by a negative feedback loop between MDM2 and p53. MDM2 up-regulates p53 transcription activity directly, and leads to p53 nuclear export and proteasome mediated degradation. As such, elevation of p53 up-regulates MDM2 and subsequently, p53 is down-regulated[5]. DNA damage and oxidative stress are the main stimuli of p53 protein expression. Oxidative and genotoxic stress activate PI3 kinase pathways at DNA break sites and these kinases phosphorylate MDM2 and p53, leading to p53 stabilization and activation of p53 pathways[6-8]. MDM2 expression is also regulated by specificity protein 1 (Sp1) that binds to GC-rich motifs of many promoters and is involved in many cellular processes, including cell differentiation, cell growth, apoptosis, immune responses and DNA damage[9]. Sp1 is induced by oxidative stress and regulates expression of vascular endothelial growth factor A (VEGF-A)[10], which plays a critical role in pulmonary vascular remodeling and emphysema[11,12].

In addition to inhibiting proliferation, p53 may promote differentiation. The role of p53 as a tumor suppressor is generally attributed to a stop in proliferation of precancerous cells through the induction of cell-cycle arrest or apoptosis, but p53 also has essential functions in embryonic development and differentiation control[13]. Most of the biological and physiological functions of p53 have been examined by genetically modified mice. Because of p53’s critical effects on cell proliferation and apoptosis, p53 deficient mice were estimated to have severe developmental disturbances. Interestingly, the p53 deficient mice appeared with no birth defects, however, most of the mutant mice largely developed tumors such as lymphomas around the age of 6 mo[13,14]. This implies that basal levels of p53 expression may regulate normal cell growth and development, and that a further reduction in p53 has no significant effects on p53-dependent pathways[15]. Another possibility is that when p53 is lacking, its function in tissue development is substituted by the function of p63 and p73[16]. Conversely, higher expressed p53 induced by genotoxic stress, could play a critical role in cell growth and development.

In recent years, microRNAs (miRNAs) which were modulated by p53 was found by several groups[17,18]. miRNAs, known as non-coding RNA that down-regulate gene expression by bind to target mRNAs at the region of complementary sequences in the code or the 3’-untranslated sequences. Several miRNAs are involved in the regulation of proliferation, differentiation and apoptosis of the cells[19,20]. miR34a is the best-studied miRNA induced by p53, and its expression is highly associated with apoptosis and cell cycle control in cancer cells[21]. We have previously reported that miRNA34a is also related to the expression of hypoxia-inducible factor-1 alpha (HIF-1α) both in the lung tissues from animal and human[22-24], which means miRNA regulated by p53 is also involving in the lung tissue remodeling via p53 pathway. The present article will continue with a review of in vitro and in vivo studies on lung tissue remodeling especially focused on the p53 pathways (Figure 1). In addition, we will discuss recently published strategies designed to identify and characterize novel function of p53.

Figure 1 Schematic illustration of regulation and pathway of p53. Genotoxic stress or oxidative stress induce phosphorylation of mouse double minute 2 homolog (MDM2) and p53 protein and activate p53 pathway, which induce cell cycle arrest and/or apoptosis. Puma: P53 up-regulated modulator of apoptosis; Bax: Bcl-2-associated X protein.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE

Chronic obstructive pulmonary disease (COPD) is a heterogeneous and multi-component disease comprising of a combination of small airways disease and parenchyma destruction, and is characterized by airflow limitation. The two characteristic features of COPD are chronic bronchitis and emphysema and each is associated with a specific physiopathology and set of symptoms[25]. In the clinical situation, the presence of emphysema can be expected on the basis of medical history, physical examination and pulmonary function tests, but since emphysema is defined as a structural pulmonary abnormality, its presence can only be confirmed by CT imaging or lung histology[26].

Both higher rates of apoptosis[27,28] and increased alveolar cell proliferation[29] have been reported in the human emphysematous lung, suggesting that a high cell turnover and perhaps impaired regeneration processes of the lung cell may be critical in the pathogenesis of emphysema. In emphysema patients, cigarette smoke will cause exposure of large amounts of free radicals including superoxide (O^2-), hydroxyl radical and hydrogen peroxide (H₂O₂)[30]. Even after cessation of smoking, oxidative stress comes from activated macrophages and neutrophils, which continue to be present in emphysematous lung[31,32]. Probably related to the increased oxidative stress[33], increased level of p53 have been reported in emphysematous lung tissue and the p53 protein expression is significantly higher in lung tissue from patients with emphysema secondary to smoking when compared with tissue from smokers without emphysema or non-smokers[24,34,35], suggesting a possible direct relationship between p53 expression and oxidative stress from smoking.

It can be hypothesized that the susceptibility of smokers to develop COPD is at least in part related to polymorphisms in genes coding for p53 and p53 related proteins. Lee et al[36] reported that polymorphisms in the p53 codon 72 (rs2279744) and the p21 codon 31 (rs1801270) were significantly associated with the occurrence of smoking-related COPD in Taiwan Chinese patients. The p53 codon72 encodes either a proline or arginine residue and resides in a proline-rich region which is important for the function of p53, especially its ability to induce apoptosis. Some studies suggest that cells from individuals with the proline allele of p53 codon72 will undergo less apoptosis in response to genotoxic stress compared with individuals with the arginine allele of p53 codon72[37,38].

The second component of COPD: airway narrowing due to chronic bronchitis may also be affected by the p53 pathway through the induction of bronchial cell apoptosis and/or proliferation in response to the oxidative stress from smoking[39,40]. p53-dependent cell death is caspase dependent[41], whereas p21 does not induce cell death on its own[42]. Although p21 regulates cell-cycle arrest in response to p53 activation[43], previous reports on p21 polymorphism showed conflicting reports linking p21 overexpression to both cancer suppressive as well as promoting effects[44]. Thus, each of these observations remains controversial, especially in the pulmonary emphysema due to lung cell apoptosis (Figure 2).

Figure 2 The hypothetical role of p53 in chronic obstructive pulmonary disease. Oxidative stress from smoking activates the p53 pathway and thereby induces pulmonary emphysema through apoptosis of alveolar cells and vascular endothelial cells. The p53 pathway could be involved in bronchial remodeling.

BRONCHIAL ASTHMA

Epithelial repair processes play an important pathogenic role in initiating and maintaining the airway inflammation and remodeling of asthma. Epithelial loss has been described as a typical pathologic feature of asthma[45,46]. Several studies on lung cells and in animal suggested that increased presence of apoptotic cells in the asthmatic bronchus contributes to the pathogenesis of chronic inflammation and remodeling[45]. Defects in apoptotic cell uptake by alveolar macrophages (defective efferocytosis) could contribute to the chronic inflammation of asthmatic lungs[47]. The p53 pathway could be involved in the persistent presence of apoptosis cells in the asthmatic bronchus. Saccucci et al[48], showed by polymorphism analysis that asthmatic children exhibited a higher frequency of the arginine genotype in p53 codon 72 than the proline genotype. This result is in line with a previous report showing the increased presence of apoptosis cells in the asthmatic bronchus, since the arginine genotype of p53 codon 72 is associated with a higher induction of apoptosis compared to the proline genotype[37,38]. Interestingly, the polymorphism pattern in asthma is completely opposite from the pattern in COPD[36].

Mast cells play a central role in the allergic asthmatic response. Activation of mast cells via cross-linking of allergen-specific IgE induces degranulation and release of mediators, particularly histamine and lipid mediators, chemokines, and cytokines that evoke the different symptoms of the asthmatic response[49]. Interestingly, Suzuki et al[50], reported that p53 suppresses IgE mediated mast cell activation through NF-κB mediated cytokine production. Another study showed that p53 plays a pivotal role in mast cell survival via mTOR pathways[51]. These results suggest that p53 activation by genotoxic stress in the bronchus from asthmatic patients may prevent progression of bronchial remodeling by inhibiting mast cell and IgE mediated inflammation. Together these results may imply that increased p53 activity may promote disease susceptibility but, at the same time, attenuate the progression of bronchial inflammation and/or remodeling. Longitudinal follow-up of patients having different p53 gene pathways will be necessary to confirm the genomic role on the disease.

PULMONARY HYPERTENSION

Pulmonary hypertension is defined clinically as a mean pulmonary artery pressure of greater than 25 mmHg. In most mammals, chronic hypoxia causes chronic pulmonary hypertension. Hypoxic environment is known as one of the key factors to induce pulmonary vascular remodeling and secondary pulmonary hypertension[52]. According to a WHO statement in 1996, approximately 140 million people were living at high altitudes more than 2500m and some of residents were living at altitudes more than 4000 m. After a couple of weeks living at such high altitude, lowlanders manifest secondary pulmonary hypertension, which cannot recover completely by oxygenation[53], indicating manifestation of chronic hypoxia causes chronic pulmonary hypertension and pulmonary vascular remodeling[54]. Remodeling of small pulmonary vessels by proliferation of pulmonary vascular cells features secondary pulmonary hypertension[55-57]. These phenomena indicate enhancement of pulmonary vascular cell proliferation induced by hypoxic environment make a considerable contribution to the progression of hypoxic pulmonary vascular remodeling and chronichypoxicpul monaryhypertension (CHPH).

Induction of the p53 induces the expression of CDK inhibitor p21, which plays a critical role in the proliferation of pulmonary arterial smooth muscle cells[43,58]. The p21 has been thought to a pivotal regulative factor of cell cycle in cells exposed to hypoxic environment and oxidative stress[59-61]. There are many cells undergoing apoptosis in tumors with wild-type p53 gene exposed to hypoxic environment, whereas tumors with mutational inactivated p53 gene have reduced levels of apoptosis in hypoxic regions[62]. Embryo fibroblasts from p53 knockout mouse are relatively resistant to apoptosis induced by hypoxia, and have better proliferative properties compared to the cells with wild-type p53 gene[63]. Hypoxic p53 accumulation is closely correlated with HIF-1α, known as a transcriptional factor which plays a central role in the control of angiogenesis during hypoxia[64,65]. Previously, we reported that hypoxic p53 accumulation is relevant to HIF-1α expression in lung tissue and pulmonary vascular cells[22,66]. It was shown that chronic hypoxia was associated with increases in p53 and p21 expression in wild-type mice, whereas the mutation of the p53 gene attenuated the hypoxia induced p21 expression and resulted in more severe vascular remodeling of small pulmonary arteries[22]. Furthermore, Mouraret et al[67], reported that the activation of p53 by nutlin-3a, known as inducer of p53 protein by inhibition of p53-MDM2 binding, prevented CHPH in mice[67]. These findings suggest that under hypoxic environment, induced p53 and the p53 signaling pathway play a role as a negative feedback loop to attenuate excessive proliferation of vascular cells and pulmonary arterial remodeling, and serve as a central role as a regulator of hypoxic vascular remodeling of small pulmonary artery.

p53 mutations or defects in cancer cells can enhance interleukin 6 (IL-6) expression[68]. IL-6 overexpression is known to change the balance between pro- and anti-apoptotic proteins, which can promote vascular remodeling and pulmonary hypertension[69]. We previously showed that the endothelial cell IL-6 expression in pulmonary arteries was increased in a non-hypoxic animal model of pulmonary hypertension[70]. The results could be explained by activation of a IL-6/STAT3/HIF-1α signaling axis, as was previously described by Nilsson et al[71], or involvement of NF-κB and HIF-1α[72]. Recently, Dickinson et al[73], showed that down-regulation of early growth response protein 1 (Egr-1) resulted in a reduction of pulmonary vascular proliferation and increased apoptosis with decreases of IL-6 and p53 expression in rats[73]. The Egr-1 regulation of p53 and IL-6 in vascular remodeling could be another of potential target of CHPH treatment in future (Figure 3).

Figure 3 Possible mechanistic roles of p53 in pulmonary hypertension. Hypoxia induces vascular proliferation via hypoxia-inducible factor-1α (HIF-1α) activation, which may be negatively regulated by p53 activation and also influence vascular remodeling induced by interleukin 6 (IL-6).

CONCLUSION

In normal cells, the expressed level of p53 is very low, and the biochemical effects of p53 are thought to be latent. In response to DNA damage, oxidative stress and hypoxia, p53 can become stabilized and activates p53 pathways. Previously, the activation of p53 was believed to be primarily involved in the prevention of propagation of cells with potentially dangerous genetic lesions[74], as a guardian of genome. However, a number of studies have suggested that the effects of p53, including arresting cell cycle and triggering apoptotic cell death, are also involved in lung development and remodeling. Because hypoxia and oxidative stress are environmental factors closely related with CHPH in highlanders, and COPD, asthma and asthma COPD overlap syndrome in smokers, the impact of p53 activity on pulmonary disease has been shown to be much larger. Genomic analysis including genome-wide association studies (GWAS) is a common tool for the analysis of disease susceptibility and profiles in pulmonary diseases[75] and has shown its use in the detection of disease responsible genes and disease specific features. Such studies have shown interesting data pertaining to p53 polymorphisms, but the data is still conflicting at times. The regulatory actions p53 with respect to proliferation and apoptosis are complex and so are the processes involved in lung and vascular remodeling, where numerous interactions exist between vascular endothelial cells, smooth muscle cells, fibroblast and epithelial cells. For instance, hypoxic-ischemic states could release mitogenic factors from pulmonary vascular endothelial cells and adventitial fibroblasts around smooth muscle cells, resulting pulmonary vascular smooth muscle proliferation. Conversely, pulmonary vascular endothelial cells damaged by hypoxia may be able to decrease the expression of suppressors of smooth muscle proliferation. Furthermore, hypoxic proliferation of lung fibroblasts may lead to the secretion of matrix proteins that play a critical role for the smooth muscle cells proliferation[76]. The two characteristic features of COPD, pulmonary emphysema and chronic bronchitis, may come about through the interactions between cigarette smoke exposure and different p53 genotypes. We assume that a deeper understanding of pathophysiologic mechanisms including characterization of genotypes and phenotypes will make therapeutic advances and improved diagnosis.

ACKNOWLEDGMENTS

We wish to thank Professor Norbert F Voelkel, Virginia Commonwealth University, Richmond, VA, United States for his critical reading of this manuscript.