Arginine is a key player in fibroblasts during the course of IPF development

Main Text

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by the activation of invasive fibroblasts, which are involved in the uncontrolled production and deposition of extracellular matrix (ECM) in the lung parenchyma. No therapeutic approach is, thus far, available to halt or reverse the process of IPF, although nintedanib and pirfenidone, two US Food and Drug Administration (FDA)-approved drugs, have shown that it is possible to slow the decline of lung function without a noticeable effect upon overall mortality. Importantly, the presence of severe side effects can result in drug discontinuation. As a result, the median survival time of patients with IPF is only 2–3 years from diagnosis, and the 5-year mortality rate still ranges from 70% to 80%.¹ Therefore, there is a need for the identification of a safer and more effective therapeutic target against IPF. In this issue of Molecular Therapy, Li et al.² provide experimental evidence that argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme responsible for the biosynthesis of the endogenous semi-essential amino acid arginine in the urea cycle, is lost in fibroblasts derived from IPF patients. They further show that deprivation of exogenous arginine attenuates fibroblast proliferation, migration, and invasion, thereby protecting mice against bleomycin (BLM)-induced pulmonary fibrosis.²

Metabolic reprogramming and dysregulation are involved in IPF pathogenesis.³ Metabolomic abnormalities in alveolar epithelial cells, macrophages, fibroblasts, and myofibroblasts contribute to the uncontrolled ECM synthesis and destruction of alveolar architecture. Transforming growth factor β (TGF-β), a well-known fibrotic factor, enhances 6-phosphofructo-2-kinase/fructose-2,6-biosphosphatase 3 (PFKFB3) expression along with the generation of fructose 2,6 bisphosphate, an important product of glycolysis.³ Glycolysis, in turn, stabilizes the hypoxia-inducible factor 1α (HIF-1α) to promote fibroblast activation, thereby exacerbating pulmonary fibrosis.³ Dysregulated fatty acid oxidation also predisposes individuals to IPF development. Inhibition of acetylCoA carboxylases, the rate-limiting enzymes of fatty acid metabolism, represses TGF-β-induced collagen synthesis by 65% in lung fibroblasts.³ Moreover, fatty acid oxidation enhances macrophage M2 polarization, which then induces IPF development and progression.⁴ Similarly, altered amino acid metabolism induces a profibrotic cellular phenotype. Particularly, arginine is involved in collagen synthesis, cell apoptosis, and ammonia removal (Figure 1).³ High levels of arginine metabolites, including creatine, putrescine, spermidine, 4-hydroxyproline, and proline-hydroxyproline dipeptide, are detected in IPF patients. L-proline, a precursor of collagen, is synthesized from L-arginine by the actions of arginases, while Arginase-1, an M2 macrophage marker, is highly expressed in alveolar macrophages from IPF patients,⁵ suggesting that M2 macrophages increase L-proline to promote synthesis of collagen by myofibroblasts. In support of this notion, inhibition of Arginase-1 diminishes collagen deposition and improves BLM-induced pulmonary fibrosis.³

Figure 1.

Schematic representation of metabolism of arginine in the pathogenesis of idiopathic pulmonary fibrosis (IPF)

IPF is characterized by the presence of high levels of arginine and its metabolites in the circulation. The metabolites, in turn, worsen IPF at least by induction of apoptosis of alveolar epithelial type II cells and activation of fibroblasts, coupled with ECM production and deposition in the lung parenchyma. Given that ASS1 serves as a rate-limiting enzyme for biosynthesis of the endogenous semi-essential amino acid arginine in the urea cycle, fibroblasts derived from IPF patients manifest ASS1 deficiency, which might be a compensatory regulatory mechanism to control the homeostatic endogenous arginine levels. However, food intake is still the main source of arginine. Therefore, deprivation of exogenous arginine may be a viable strategy against IPF.

Given that ASS1 is a rate-limiting enzyme that catalyzes arginine biosynthesis in the urea cycle, it is considered a potent tumor suppressor by regulating a variety of biological processes.⁶ Notably, ASS1 is generally highly expressed in normal tissues but lost in a range of tumor cells.⁶ Therefore, eliminating extracellular arginine primes tumor cells to undergo apoptosis in arginine auxotrophs. Excitingly, several clinical trials of arginine-lowering agents, such as pegylated arginine deiminase (ADI-PEG20), have provided encouraging evidence of clinical benefit and low toxicity in patients with ASS1-negative tumors. Interestingly, similar to cancer, IPF could be thought of as a neoproliferative disorder in the lung, as evidenced by the analogous pathways involved in disease progression and anomalous fibroblast behaviors (e.g., uncontrolled proliferation, disturbed cell-to-cell communication, and resistance to apoptosis). However, the expression of ASS1 and its contribution in IPF has not been established.

Li et al.² now demonstrate that ASS1 expression is significantly reduced in lung sections from IPF patients. Indeed, ASS1 was nearly absent in fibrotic foci. Importantly, fibroblasts from the lungs of IPF patients exhibited a pattern of ASS1 expression similar to that observed in cancer cells.² However, the mechanisms behind the loss of ASS1 expression in fibroblasts derived from IPF patients are complex, and may include: (1) ASS1 promoter hypermethylation;⁶ (2) interplay between c-Myc and HIF-1α;⁶ (3) repressed ASS1 translation by microRNA (miRNA; e.g., miR-1291);⁷ and (4) transactivation by p53 in response to genotoxic stress.⁸ In fact, altered DNA methylation, overexpression of c-Myc and HIF-1α, and attenuated p53 expression have been observed in fibroblasts from IPF lungs or mouse lungs following BLM-induced fibrosis.^4,9,10 Since ASS1 deficiency is a typical feature of activated proliferating fibroblasts—coupled with sustained collagen deposition during the course of IPF development—future studies should address the potential involvement of the above factors in fibroblast ASS1 deficiency. Importantly, fibroblasts from IPF patients are vulnerable to arginine deprivation, as evidenced by the attenuated proliferation during growth in arginine-free conditions or following ADI treatment—conditions that do not adversely affect normal fibroblasts.² In line with this observation, arginine deprivation increases nintedanib efficacy and protects mice from BLM-induced pulmonary fibrosis. Particularly, deficiency of the arginine transporter solute carrier family three member two (SLC3A2) in fibroblasts represses ECM production following TGF-β treatment.¹¹ Collectively, high levels of arginine are observed in the fibrotic microenvironment, which contributes to IPF initiation and progression. As a result, the manifestation of ASS1 deficiency in fibroblasts during IPF development could represent a compensatory feedback regulatory mechanism to control homeostatic endogenous arginine levels. Since arginine is a semi-essential amino acid, exogenous intake remains the major source of arginine for the body. Therefore, fine control of extracellular arginine levels in the fibroblast microenvironment could be a viable strategy against IPF in clinical settings.

Although the findings reported by Li et al.² are exciting, a number of questions remain to be addressed, such as the impact of arginine on fibroblast-to-myofibroblast transition. Moreover, long-term studies in larger animal models are needed to ascertain whether arginine deprivation is associated with major side effects in IPF patients. In summary, deprivation of exogenous arginine exhibits a notable suppressive effect on pulmonary fibrosis and, as such, this ASS1 metabolite has emerged as a key player in fibroblast proliferation, migration, and invasion with the potential to be a key target against IPF in clinical settings.