Niche-Dependent Regulation of Lkb1 in the Proliferation of Lung Epithelial Progenitor Cells

Lung homeostasis and regeneration depend on lung epithelial progenitor cells. Lkb1 (Liver Kinase B1) has known roles in the differentiation of airway epithelial cells during embryonic development. However, the effects of Lkb1 in adult lung epithelial progenitor cell regeneration and its mechanisms of action have not been determined. In this study, we investigated the mechanism by which Lkb1 regulates lung epithelial progenitor cell regeneration. Organoid culture showed that loss of Lkb1 significantly reduced the proliferation of club cells and alveolar type 2 (AT2) cells in vitro. In the absence of Lkb1, there is a slower recovery rate of the damaged airway epithelium in naphthalene-induced airway epithelial injury and impaired expression of surfactant protein C during bleomycin-induced alveolar epithelial damage. Moreover, the expression of autophagy-related genes was reduced in club cells and increased in AT2 cells, but the expression of Claudin-18 was obviously reduced in AT2 cells after Lkb1 knockdown. On the whole, our findings indicated that Lkb1 may promote the proliferation of lung epithelial progenitor cells via a niche-dependent pathway and is required for the repair of the damaged lung epithelium.


Introduction
The lung epithelium protects the lungs from environmental insults. The maintenance of lung tissue homeostasis and regeneration depend on healthy lung epithelial progenitor cells [1]. Two major types of epithelial progenitor cells are responsible for regenerating the airway and alveolar epithelial cells during lung injury. Club cells, as airway progenitor cells, can proliferate and differentiate into goblet and ciliated cells [2] at a steady state or after airway epithelial injury. Alveolar type 2 cells (AT2) synthesize and secrete numerous surfactant proteins that reduce alveolar surface tension and have antioxidant and antibacterial effects [3][4][5]. As alveolar progenitor cells, AT2 cells are capable of self-renewing and generating alveolar type 1 (AT1) cells at a steady state or after bleomycin (BLM)-induced lung injury [6][7][8]. Abnormal lung epithelial progenitor cell function leads to many respiratory diseases. Therefore, the regeneration of club and AT2 cells must be tightly regulated during lung homeostasis and damage.
Liver kinase B1 (LKB1), a tumor suppressor encoded by serine/threonine kinase 11 (Stk11), was first associated with Peutz-Jeghers syndrome and was involved in the control of embryonic development, tissue homeostasis, stem cell function, energy metabolism, and apoptosis [9][10][11][12][13][14][15][16][17][18]. The constitutive deficiency of Lkb1 causes embryonic lethality and, 2 of 14 in adult mice, contributes to weight loss and disrupted metabolism [16]. The regenerative capacity of hematopoietic stem cells in the bone marrow of irradiated mice decreases significantly after Lkb1 deletion [7]. The loss of Lkb1 in periosteal mesenchymal progenitor cells induces osteoblastogenesis by activating the mammalian target of rapamycin complex 1 (mTORC1) [19]. The loss of Lkb1 in intestinal epithelial cells leads to impaired immune barriers and increased bacterial susceptibility [14]. Lkb1 knockout in the renal epithelium activates chemokines and recruits inflammatory cells, leading to polycystic kidney disease [20]. Lkb1 deletion promotes tumor cell proliferation by regulating mitogenactivated protein kinase (MAPK) signaling [21] and induces metabolic reprogramming [18] to promote tumorigenesis and lung cancer progression. Furthermore, Lkb1 is required for the normal ciliated cell differentiation in both embryonic and adult lungs [10]. Our previous research has shown that Lkb1 deficiency in the embryonic lung epithelium results in weight loss and tamoxifen-induced death within 5 weeks. Lkb1 regulates airway goblet cell metaplasia by mediating interactions between airway progenitors and macrophages [22].
In conclusion, our results suggested that Lkb1 may promote club cell proliferation via an autophagy-dependent pathway, while promoting AT2 cell proliferation in an autophagyindependent manner. Taken together, our results proposed a niche-dependent regulatory mechanism by which Lkb1 may affect airway and alveolar progenitor cell proliferation during homeostasis and lung injury.

Distribution and Expression Levels of Lkb1 in Human and Mouse Lung Tissues
We have previously observed that the loss of Lkb1 in the embryonic lung disrupts the lung epithelial structure and decreases the epithelial cell abundance in the adult lung [22]. Here, we investigated the mechanism by which Lkb1 regulates the proliferation of lung epithelial progenitor cells in adult mice. Based on comprehensive analyses of published single-cell transcriptome sequencing data (GSE122960, GSE128033, GSE135893, and GSE136831) [23][24][25][26], we detected LKB1 expression in various cell populations (epithelial, mesenchymal, endothelial, and immune cells) in normal human lung tissues ( Figure 1A-C). We further observed the distribution and expression of LKB1 in lung epithelial cells (AT1, AT2, club, goblet, and ciliated cells) ( Figure 1D-F). Similarly, we analyzed the distribution and expression of Lkb1 in different cell types in mouse lung tissues, including lung epithelial cells ( Figure 1G-L). These results suggested that Lkb1 is widely distributed in lung tissues and a certain expression level is maintained in lung epithelial cells at a steady state.

Lkb1 Is Required for Lung Epithelial Progenitor Cell Proliferation In Vitro
As the main lung epithelial progenitor cells, club and AT2 cells are involved in the maintenance of lung epithelium function and repair after injury. Therefore, we constructed mice with a conditional deletion of Lkb1 in airway club cells or alveolar AT2 cells to investigate the function of Lkb1 in the regeneration of lung epithelial progenitor cells in adult mice. We created Scgb1a1 CreER ; Lkb1 f/f mice by crossing Lkb1 f/f mice with Scgb1a1 CreER mice, in which Lkb1 was conditionally deleted in airway club cells (Figure 2A). After the administration of tamoxifen to induce the loss of Lkb1 in club cells, we did not detect obvious histological abnormalities in adult mice at a steady state ( Figure 2B,C). To further evaluate the effect of Lkb1 on the proliferation of club cells in vitro, we used an FACS-based method to separate mouse club cells ( Figure 2D). Lin − EpCAM + Sca1 + CD24 + club cells were sorted into organoid cultures. We observed no significant differences in the proportion of epithelial cells and abundance of club cells in the total live cell population after Lkb1 deletion by flow cytometry ( Figure 2E). Organoid culture indicated that the colony forming efficiency (CFE) (3.960 ± 0.779 (Lkb1 KO) vs. 10.873 ± 3.261 (Control), p = 0.001) and colony size (146.200 ± 13.580 (Lkb1 KO) vs. 175.591 ± 11.076 (Control), p = 0.003) of club cells sorted from Scgb1a1 CreER ; Lkb1 f/f mice were obviously decreased ( Figure 2F-I). These results suggested that Lkb1 is essential for the proliferation of mouse club cells in vitro.

Lkb1 Is Required for Lung Epithelial Progenitor Cell Proliferation In Vitro
As the main lung epithelial progenitor cells, club and AT2 cells are involved in the maintenance of lung epithelium function and repair after injury. Therefore, we the proportion of epithelial cells and abundance of club cells in the total live cell population after Lkb1 deletion by flow cytometry ( Figure 2E). Organoid culture indicated that the colony forming efficiency (CFE) (3.960 ± 0.779 (Lkb1 KO) vs. 10.873 ± 3.261 (Control), p = 0.001) and colony size (146.200 ± 13.580 (Lkb1 KO) vs. 175.591 ± 11.076 (Control), p = 0.003) of club cells sorted from Scgb1a1 CreER ; Lkb1 f/f mice were obviously decreased ( Figure 2F-I). These results suggested that Lkb1 is essential for the proliferation of mouse club cells in vitro.  To explore the effect of Lkb1 on AT2 cell proliferation, we generated Sftpc CreER ; Lkb1 f/f mice with conditional loss of Lkb1 in alveolar AT2 cells ( Figure 3A). Tamoxifen was administered to mice aged 8-12 weeks by intraperitoneal injection to induce Lkb1 deletion in alveolar AT2 cells. The lungs of Sftpc CreER ; Lkb1 f/f mice showed no significant histological abnormalities at a steady state ( Figure 3B,C). To further analyze the effect of Lkb1 on the proliferation of AT2 cells in vitro, we isolated AT2 cells from mouse lung tissues by FACS. The proportion of epithelial cells and the ratio of AT2 cells to total live cells did not differ between wild-type mice and mice with Lkb1 deletion ( Figure 3E). The CFE (0.637 ± 0.009 (Lkb1 KO) vs. 1.050 ± 0.020 (Control), p = 0.001) and colony size (101.806 ± 36.761 (Lkb1 KO) vs. 122.730 ± 39.257 (Control), p = 0.001) were reduced in the deletion of Lkb1 ( Figure 3D-H). Consistent with this, immunofluorescence staining of colonies showed that the fraction of Ki67 + pro-SPC + cells over total pro-SPC + cells was lower (6.050 ± 0.522 (Lkb1 KO) vs. 29.302 ± 7.109 (Control), p = 0.007) that in the absence of Lkb1 ( Figure 3I,J). Collectively, these results suggested that Lkb1 is indispensable for mouse AT2 cell proliferation in vitro. Considering that the deletion of Lkb1 may lead to the death of AT2 cells, we observed AT2 cell viability during organoid culture, and the results indicated that AT2 cells still survive (GFP + cells), but the GFP + cells could not proliferate normally to form clones after Lkb1 deletion (Supplementary Figure S1A,B). On the other hand, bronchoalveolar lavage fluid (BALF) was added to the organoid cultures to evaluate the effect of growth factors in microenvironment on alveolar epithelial progenitor cell proliferation in vitro. Our results showed that reduced proliferation ability of AT2 cells caused by Lkb1 deletion could not be rescued by BALF supplementation in organoid culture (Supplementary Figure S2A  Five independent experiments were conducted (n = 5). All data are presented as means ± SD, ** p < 0.01; *** p < 0.001.

Lkb1 Is Beneficial for the Recovery of Lung Epithelium after Injury
Furthermore, we explored the regeneration of lung epithelial progenitor cells after Lkb1 deficiency during lung injuries in vivo. In both Lkb1 f/f mice and Scgb1a1 CreER ; Lkb1 f/f mice, 250 mg/kg naphthalene was injected intraperitoneally to induce airway epithelium injury, and lung tissues were collected on days 0, 2, and 20 after naphthalene administration ( Figure 4A). The decrease in club cells and loss of body weight in Scgb1a1 CreER ; Lkb1 f/f mice caused by naphthalene were more remarkable than those in Lkb1 f/f mice ( Figure 4B). Immunofluorescence staining of lung tissues for the detection of Cyp2f2 showed a more severe airway injury and slower the rate of recovery of damaged airways in the case of the deletion of Lkb1 ( Figure 4C). The above results suggested that Lkb1 may protect the lungs from naphthalene-induced damage and promote the repair of the damaged mouse airway epithelium. mice, 250 mg/kg naphthalene was injected intraperitoneally to induce airway epithelium injury, and lung tissues were collected on days 0, 2, and 20 after naphthalene administration ( Figure 4A). The decrease in club cells and loss of body weight in Scgb1a1 CreER ; Lkb1 f/f mice caused by naphthalene were more remarkable than those in Lkb1 f/f mice ( Figure 4B). Immunofluorescence staining of lung tissues for the detection of Cyp2f2 showed a more severe airway injury and slower the rate of recovery of damaged airways in the case of the deletion of Lkb1 ( Figure 4C). The above results suggested that Lkb1 may protect the lungs from naphthalene-induced damage and promote the repair of the damaged mouse airway epithelium.  Similarly, 2 U/kg Bleomycin (BLM) was injected intratracheally to induce alveolar epithelium injury in Lkb1 f/f mice and Sftpc CreER ; Lkb1 f/f mice, and lung tissues were collected on day 14 after BLM administration ( Figure 4D). The body weight loss and degree of pulmonary fibrosis did not differ between the two groups ( Figure 4E,F). There were no significant differences in the expression levels of pulmonary fibrosis markers, including fibronectin (Fn), Col1α, and α-SMA, between mice with and without Lkb1 ( Figure 4G). Immunofluorescence staining showed normal expression levels of ABCA3 in lung tissue between wild-type mice and mice with Lkb1 deletion, while the proliferation of AT2 cells and the expression of SPC were impaired in the lung tissue of Sftpc CreER ; Lkb1 f/f mice ( Figure 4H). These results showed that the secretion of SPC from AT2 cells was normal, but the expression of SPC was impaired after Lkb1 deletion. All the data suggested that Lkb1 may be beneficial for the recovery of the mouse lung epithelium after lung injury.
Claudins, as tight junction proteins, contribute to the proliferation and tumorigenesis of lung progenitor cells. Claudin-18, mostly expressed in alveolar epithelial cells [30,31], is rarely expressed in airways. We did not detect a change in the expression of Claudin-18 in lung tissues, but observed decreased levels in both AT2 cells (0.396 ± 0.052 (Lkb1 KO) vs. 0.512 ± 0.026 (Control), p = 0.004) and organoid cultures (0.060 ± 0.044 (Lkb1 KO) vs. 0.201 ± 0.097 (Control), p = 0.023) after Lkb1 deficiency ( Figure 5C-E). Therefore, we hypothesized that Claudin-18 may be involved in Lkb1 knockout-induced reduction in the proliferation of mouse AT2 cells through affecting cell-cell adhesion/integrity in vitro. These findings suggested that Lkb1 may promote the proliferation of mouse club cells and AT2 cells via different mechanism ( Figure 5F).

Discussion
The pulmonary epithelium plays an essential role in gas exchange and host defense. As the dominant lung epithelial progenitor cells, club cells, and AT2 cells are responsible for maintaining homeostasis and repairing injuries [1,28]. In this study, we found that Lkb1 is indispensable for the proliferation of normal lung epithelial progenitor cells in adult mice at a steady state. The loss of Lkb1 significantly restricted the proliferation of club cells and AT2 cells, as validated by organoid culture in vitro. Additionally, in the absence of Lkb1, the recovery rate of the damaged airway epithelium was slower, and the secretion of SPC from AT2 was impaired. Furthermore, we observed altered expression levels of autophagy markers and Claudin-18 in the mouse pulmonary epithelium with an Lkb1 deficiency. Our results suggested that Lkb1 may promote club cell proliferation via an autophagy pathway, but promote AT2 cell proliferation via Claudin-18. Our research will provide important insights into the mechanism of clinical treatment of Lkb1-related lung diseases.
Lkb1, a tumor suppressor, is strongly expressed in both human and mouse lung tissues. It is involved in the occurrence and progression of numerous diseases, especially in lung adenocarcinoma [32,33]. Tissue-specific Lkb1 knockout studies have suggested that Lkb1 contributes to stem cell regeneration and tissue homeostasis [19,[34][35][36]. Lkb1 directly activates MAPK to regulate cell survival, and Lkb1-deficient cells are highly sensitive to apoptosis caused by energy stress [17]. Lkb1 deletion disrupts cell polarity and promotes collagen remodeling during tumor invasion [13]. Tang et al. demonstrated that the Lkb1/MARK3/ERK1/2 signaling cascade is a crucial regulator of ciliated cell fate and multiciliogenesis [10]. Our previous study revealed that Lkb1 loss upregulates RELM-α in club cells, thereby regulating goblet cell differentiation metaplasia [22].
Autophagy is a conserved cellular process that maintains the regenerative potential of the epithelium in response to stress [27]. Autophagy affects the regenerative and therapeutic potential of mesenchymal stem cells [37]. Hematopoietic stem cells rely on autophagy to maintain normal metabolism and functions [38]. Aging muscle stem cells promote muscle regeneration via autophagy [39,40]. Hair follicle stem cells depend on autophagy to maintain their differentiation capacity [41]. Mice rely on autophagy to reduce excessive reactive oxygen species (ROS) and maintain the regeneration of intestinal stem cells [42]. Autophagy has been demonstrated to promote the regeneration of the airway epithelium and alveolar epithelium by metabolic reprogramming during pulmonary injury [1,28,29]. Similarly, our results suggested that Lkb1 promotes club cell proliferation in an autophagydependent pathway, and the recovery rate of the damaged airway epithelium was slower in the absence of Lkb1 in airway progenitor cells. The loss of Lkb1 in AT2 cells decreased the expression of got associated with glutamine metabolism, which is required for alveolar regeneration during lung injury.
A growing body of research indicates that Claudins are involved in the regulation of cell proliferation and polarity. Claudins are tight junction proteins with a significant effect in lung progenitor cell proliferation and tumorigenesis [30]. Claudin-18 is mainly expressed in lung alveolar epithelial cells [30,31], and is rarely expressed in airways. Other Claudins, such as Claudin-3 and Claudin-4, are highly expressed in the airway epithelium. In mice with bleomycin-induced lung injury, the expression of genes encoding claudin proteins is reduced, especially Claudin-18 [43], consistent with our results.
Our study had several limitations. First, organoid cultures showed significantly reduced proliferation of both club cells and AT2 cells in vitro; however, we did not detect an abnormal epithelial structure under homeostasis in vivo. We speculate that a compensatory mechanism in vivo attenuates the decreased cell proliferation caused by Lkb1 deletion. Autophagy is the most likely mechanism; however, more research is needed to clearly explain the mechanism underlying the maintenance of homeostasis. Second, alveolar SPC secreted by AT2 cells reduces alveolar surface tension and increases lung compliance, thereby promoting the maintenance of a normal lung structure and function [44]. In this study, we observed a significant decrease in SPC expression after Lkb1 deficiency during BLM-induced lung injury. Although it may prevent alveolar epithelium repair, we did not observe severe lung tissue damage in the absence of Lkb1. Third, we have no direct evidence to support the hypothesis that decreased autophagy directly influences airway epithelial cell proliferation in Lkb1-deficient mice. Claudin-18, as a tight junction protein and an important regulator of lung epithelial cell proliferation [45], showed decreased expression levels in AT2 cells and organoid cultures from Lkb1-deficient mice, suggesting that decreased Claudin-18 expression may affect the organoid formation through affecting cell proliferation and cell-cell junction. However, we lack direct evidence that reduced Claudin-18 expression affects alveolar epithelial cell proliferation through cell-cell adhesion in Lkb1-knockout mice. Therefore, more works should be focused on cell-cell adhesion/integrity and further experiments are needed to verify these results.

Mice
The experimental mice were retained in a specific pathogen-free (SPF) facility at Tianjin Haihe Hospital (SYXK (Jin) 2021-0002). The mice were exposed to a 12 h light/dark cycle and had free access to food and water. Lkb1 f/f mice, originally from Dr. Ronald DePinho (Boston, MA, USA), were provided by Dr. Hongbin Ji (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China). Scgb1a1 CreER and Sftpc CreER mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). To induce Lkb1 knockdown in the airway epithelial progenitor cells, Scgb1a1 CreER mice were crossed with Lkb1 f/f mice to create Scgb1a1 CreER ; Lkb1 f/f mice. Similarly, Sftpc CreER ; Lkb1 f/f mice were created by crossing Sftpc CreER and Lkb1 f/f mice. The heterozygous mice were mated for 3-5 generations to obtain homozygous mice for experimental study. All experimental mice at 8-12 weeks of age were randomly assigned to groups. Mice were anesthetized using 1% sodium pentobarbital (50 mg/kg). All procedures involving animals were reviewed and approved by the Tianjin Haihe Hospital Animal Care and Use Committee (2021HHKT-018).

Naphthalene-Induced Airway Epithelium Injury
200 mg/kg tamoxifen (Sigma-Aldrich, St. Louis, MS, USA) was intraperitoneally injected into Lkb1 f/f and Scgb1a1 CreER ; Lkb1 f/f mice every other day for three times. After tamoxifen injection was completed, the mice rested for seven days and then were intraperitoneally injected with naphthalene (Sigma-Aldrich, St. Louis, MS, USA) dissolved in corn oil (250 mg/kg) (Sigma-Aldrich, St. Louis, MS, USA) to induce mouse airway epithelium injury. After naphthalene treatment, mouse lung tissues were collected on days 0, 2, and 20 for histological analyses.

Bleomycin-Induced Alveolar Epithelium Injury
Lkb1 f/f and Sftpc CreER ; Lkb1 f/f mice received tamoxifen (50 mg/kg, i.p.) every day for five consecutive days to induce Lkb1 knockout in mouse AT2 cells. Mice were anesthetized using 1% sodium pentobarbital and received an intratracheal injection of bleomycin (BLM) (Nippon Kayaku, Tokyo, Japan) at a dose of 2U/kg. Control animals only received phosphate-buffered saline (PBS) (Corning, Jiangsu, China). Lung tissues were separated for histological analysis on day 14 after BLM administration.

RNA Extraction and qPCR
Total RNA was extracted from lung tissues or sorted lung epithelial progenitor cells using TRIzol reagent (Invitrogen) following the manufacturer's instructions. 0.2 µg of total RNA was used for reverse transcription. Quantitative real-time PCR was performed using SYBR Green Supermix (Vazyme, China) and a Light Cycler 96 Real-Time PCR system (Roche Diagnostics, Indianapolis, IN). The PCR conditions were: 95 • C for 2 min, followed by 40 cycles of 95 • C for 10 s, 60 • C for 20 s, and 72 • C for 20 s. Gene expression was measured relative to the level of the endogenous reference gene, mouse β-actin. The primer sequences used for qPCR are shown in Table 1.

Single-Cell RNA Sequencing Analysis
The single-cell RNA sequencing data of human lung samples were downloaded from GEO database and analyzed using Seurat package on R platform. The cells expressed fewer than 3 genes, and the genes expressed in less than 200 cells were considered as low-quality cells and genes and removed from data matrices. The expression of genes was normalized using LogNormalize method: gene expression values for each cell were divided by the total number of transcripts of that cell and multiplied by 1000, and the results were then natural-log transformed using log1p. The cell types were annotated using canonical marker genes after dimension reduction and clustering based on KNN and SNN algorithm [47].

Statistical Analysis
All data are displayed as mean ± standard deviation (SD). Student's t-tests were used to evaluate differences between the experimental and control groups. P < 0.05 was considered statistically significant (* p < 0.05; ** p < 0.01; *** p < 0.001).

Conclusions
In conclusion, our findings indicated that Lkb1 may promote the proliferation of lung epithelial progenitor cells via a niche-dependent pathway, and is required for the repair of the damaged lung epithelium.  Institutional Review Board Statement: The animal study protocol was approved by the Tianjin Haihe Hospital Animal Care and Use Committee (protocol code: 2021HHKT-018).

Data Availability Statement:
The data used to support the findings of this study are included within the article.