**Cigarette Smoke Extract Stimulates MMP-2 Production in Nasal Fibroblasts via ROS**/**PI3K, Akt, and NF-**κ**B Signaling Pathways**

**Joo-Hoo Park <sup>1</sup> , Jae-Min Shin 1,2, Hyun-Woo Yang <sup>1</sup> , Tae Hoon Kim <sup>2</sup> , Seung Hoon Lee <sup>2</sup> , Heung-Man Lee <sup>2</sup> , Jae-Gu Cho 2,\* and Il-Ho Park 1,2,\***


Received: 14 July 2020; Accepted: 3 August 2020; Published: 12 August 2020

**Abstract:** Cigarette smoke exposure has been shown to be associated with chronic rhinosinusitis and tissue remodeling. The present study aimed to investigate the effects of cigarette smoke extract (CSE) on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) production in nasal fibroblasts and to determine the underlying molecular mechanisms. Primary nasal fibroblasts from six patients were isolated and cultured. After the exposure of fibroblasts to CSE, the expression levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured by real-time PCR, ELISA, and immunofluorescence staining. The enzymatic activities of MMP-2 and MMP-9 were measured by gelatin zymography. Reactive oxygen species (ROS) production was analyzed using dichloro-dihydro-fluorescein diacetate and Amplex Red assays. PI3K/Akt phosphorylation and NF-κB activation were determined by Western blotting and luciferase assay. CSE significantly increased MMP-2 expression and inhibited TIMP-2 expression but did not affect MMP-9 and TIMP-1 expression. Furthermore, CSE significantly induced ROS production. However, treatment with ROS scavengers, specific PI3K/Akt inhibitors, NF-κB inhibitor, and glucocorticosteroids significantly decreased MMP-2 expression and increased TIMP-2 expression. Our results suggest that steroids inhibit CSE-regulated MMP-2 and TIMP-2 production and activation through the ROS/ PI3K, Akt, and NF-κB signaling pathways in nasal fibroblasts. CSE may contribute to the pathogenesis of chronic rhinosinusitis by regulating MMP-2 and TIMP-2 expression.

**Keywords:** cigarette smoke extract; nasal fibroblasts; tissue inhibitor of metalloproteinases; matrix metalloproteinase; steroids

### **1. Introduction**

Chronic rhinosinusitis (CRS) is a nasal inflammatory disease with symptoms of nasal discharge/postnasal drip, nasal congestion, sinus pain/pressure, and anosmia/hyposmia lasting for at least 12 weeks. CRS is one of the most frequent chronic diseases in humans and consequently has an important socio-economic impact. Its influence on patient quality of life is even more detrimental than that of congestive heart failure, chronic obstructive pulmonary disease, and back pain [1]. CRS is a multifactorial disorder, and its pathogenesis involves interactions between environmental insults, infectious loading, and genetic predisposition. Among environmental factors, inhaled pollutants, including cigarette smoke, may play a significant role in CRS, which is a characteristic of chronic inflammatory upper airway disease [2].

Tissue remodeling is an energetic process that results in both production and degradation of the extracellular matrix (ECM) and is an important aspect in the pathogenesis of chronic inflammatory diseases in many organs. In airways, tissue remodeling is characterized by loss of epithelial integrity, goblet cell metaplasia, excessive ECM deposition, hyperplasia of mucosal cells, and basement membrane thickening. The differentiation of fibroblasts to myofibroblast is a key event in physiological and pathological tissue remodeling. Myofibroblasts are a source of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) [3]. Like other chronic inflammatory diseases of the airway, tissue remodeling is present in CRS, and obvious remodeling features differentiate the various CRS subgroups [4]. Several factors, such as MMPs, ECM, and TGF-β, are related with remodeling. Specifically, MMP-2 and MMP-9 have been associated with airway inflammatory diseases [5]. MMPs are a large family of enzymes with zinc-binding catalytic domains and are involved in the degradation of ECM components. Their extracellular activities are regulated by TIMPs. MMPs play a crucial role in various physiological processes, including tissue remodeling [6]. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are involved in ECM remodeling, which is associated with upper airway remodeling [7]. MMP-2 and MMP-9 are the principal fibroblast-derived proteinases capable of degrading substrates, including collagen, gelatin, elastin, and fibronectin.

Chronic exposure to cigarette smoke is known to be the cause of several inflammatory diseases including asthma, chronic obstructive pulmonary disease, and CRS. Cigarette smoke extract (CSE) exerts harmful effects on processes, including cell viability, adhesion, migration, and myofibroblast differentiation, in the airway. Tissue remodeling is a pathologic process believed to be related with cigarette smoke. It was previously shown that smoke can directly induce remodeling without any need for exogenous inflammatory cells in the airway [8].

CSE contains many toxic and carcinogenic chemicals, as well as unsuitable free radical that enhance reactive oxygen species (ROS) production leading to oxidative stress. ROS is known to be higher expressed in patients with CRS was higher than in control subject [9]. It damages proteins, lipids, and DNA that plays an important roles in cellular process involved in the generation and development processes of nasal polyps [10]. Airway inflammation, airway hyper-responsiveness, tissue injury, and remodeling can be induced by excessive ROS production in epithelial cells, fibroblasts and severer inflammatory cells [11,12]. Increased ROS can mediate activation of AKT and NF-κB [13]. PI3K and Akt proteins in CRS were higher than those in the control subjects [14]. The PI3K/Akt signaling pathway is an important cellular signaling that is involved in cell growth, proliferation, apoptosis, metabolism, angiogenesis, metastasis and the cellular defiance against inflammatory stimuli. Akt cascade is known to mediate ECM proteolysis [15]. NF-κB is a transcriptional factor which plays a central role in diverse cellular processes, including inflammation and immune response in airway diseases. CSE modulates variety molecular mechanisms such as ROS, PI3K/AKT and NF-κB. However, the precise mechanisms by which cigarette smoke affects airway structure and function are still under investigation.

We hypothesized that CSE-induced tissue remodeling in the upper airway is related with an imbalance between MMPs and TIMPs from nasal fibroblasts. In the present study, we aimed to determine the effects of CSE on the expression of MMPs and TIMPS and the underlying molecular mechanisms in nasal fibroblasts. We also examined the involvement of ROS, AKT, and NF-κB signaling pathways, which are known to be closely related with CSE-induced inflammation in several diseases, in these mechanisms. Additionally, we aimed to determine the effect of glucocorticoids, the first line of treatment for CRS, on the CSE-induced imbalance between MMPs/TIMPs in nasal fibroblasts.
