Interstitial lung disease (ILD) comprises many chronic lung diseases characterized by varying degrees of inflammation and fibrosis [1]. The interstitial disorder of the lung likely develops from a multifaceted interaction of genetic and environmental risk factors, aging-related events, and epigenetic profibrotic reprogramming. However, the mechanism of the progression of fibrosis remains poorly understood [2]. Pulmonary fibrosis is a clinical phenotype that reflects the end stage of chronic interstitial lung diseases characterized by progressive accumulation of extracellular matrix (ECM) in the peripheral lung, accompanied by destruction of functional alveolar gas exchange units. The most severe form of pulmonary fibrosis is idiopathic pulmonary fibrosis, a relentlessly progressive disorder that causes respiratory failure and death eventually [3].
The lung contains more than 40 different cell types [4], despite the diverse cellular composition, most of the increased ECM deposited in idiopathic pulmonary fibrosis is ascribed to the activated myofibroblasts in fibroblast foci [5]. The ECM accumulation leads to lesions that do not arise in healthy lungs but are a common feature of idiopathic pulmonary fibrosis. In clinics, the lesion numbers correlate with the survival of the patients. Many studies revealed that the lung lesions, in particular the epithelial cell injury, initiates the inflammation responses, which are categorized into different phases. During the initial injury phase, alveolar epithelial cells are activated and recruit inflammatory cells such as macrophages [6] and neutrophils [7]. Both the activated epithelial cells and immune cells release potent fibrogenic growth factors, including TGF-β, PDFG, IL-6, TNF-α, and Wnt [8], to exacerbate the pathologic process. In the next phase, these growth factors, particularly TGF-β, induce apoptosis of alveolar epithelial cells and boost activation, invasion, or apoptosis resistance of fibroblasts and myofibroblasts [9]. With the progress of the disease into the third phase, fibroblast foci, which form three-dimensional networks in lungs with usual interstitial pneumonia [10], re-dominantly occur in subepithelial regions of apoptotic or hyperplastic alveolar epithelial cells. In summary, fibrosis is characterized by an initial injury and development of subsequent remodeling of the alveolar epithelial-mesenchymal unit, which produces elevated numbers of myofibroblasts.
In the past decade, substantial progress has been made in identification of potential cellular causes of the increased myofibroblasts that occur specifically in fibroblast foci in idiopathic pulmonary fibrosis [11]. To date, several factors have been reported to be involved in the initiation and progression of pulmonary fibrosis. However, these inducers are mainly extrinsic factors such as virus infection (COVID19 [12]), aging and environmental factors, including smoking, dust inhalation, and asbestos exposure [13]. Under the challenge of these inducers, the alveolar epithelial cells are subjected to repeated micro-injury that leads to an aberrant repair response and abnormal secretion of inflammatory cytokines. These cytokines promote activation and expansion of fibroblasts/myofibroblasts, which lead to excessive ECM production and deposition [14]. As a consequence, fibroblastic foci form, with a feature of accumulated fibroblasts and myofibroblasts. To mimic this pathological process, bleomycin (BLM) injury has been widely used in the mouse model to represent all the hallmarks of human lung fibrosis. BLM induces lung damage and a transient fibrotic response with increased numbers of α-SMA+ myofibroblasts in the alveolar region [15]. The BLM induced lung injury progresses through acute injury and inflammatory phase (1–7 days post- endotracheal injection of BLM), transition phase from inflammation to active fibrosis (7–14 days post of BLM), and chronic fibrosis phase (14–21 days post of BLM) [16]. During the chronic fibrosis phase, the myofibroblast population is expanded and the deposition of ECM, such as collagens, is increased [17]. The increased deposition of lung collagen usually peaks at about 28 days post-BLM treatment [18]. Overall, BLM-induced injury boosts pathological alterations of the pulmonary fibrosis with inflammation response and activation of myofibroblasts.
CREPT, also named RPRD1B, was identified as a tumor-related protein due to its upregulation in tumors [19]. Previous studies demonstrated that deletion of CREPT impeded tumorigenesis but overexpression of CREPT promoted tumor formation. CREPT was demonstrated to upregulate cyclin D1 and cyclin B1 expression to accelerate cell cycle at both G1 and G2 phases[19, 20]. Recently, we reported that CREPT is required for the maintenance of murine intestinal stem cells as deletion of CREPT in the intestinal epithelium of mice (Vil-CREPTKO) resulted in lower body weight and slow migration of epithelial cells in the intestine [21]. We have provided evidence that CREPT participates in the regulation of intestine regeneration after irradiation and chemical-induced damage. On the other hand, we demonstrated that CREPT enhanced STAT3 transcriptional activity[22], a major regulator related to cell proliferation and inflammation responses[23].
Here, we report that CREPT, as an intrinsic factor, is required for BLM-induced pulmonary fibrosis. Deletion of CREPT attenuates BLM-induced lung fibrosis and reduces the expression of genes encoding fibrosis markers (e.g., α-SMA, FN1, COL1A1). We show that CREPT enhances fibroblast activation, proliferation, and excessive ECM production induced by TGF-β.