Persistent activation of macrophages in lungs plays a critical role in the production of matrix metalloproteinases (MMPs) that contributes to the destruction of alveolar walls, a hallmark for pulmonary emphysema. Dysregulated TGF-β1 signaling has been an essential determinant in the elevation of MMPs during the development of emphysema. Nevertheless, the mechanism for this MMP-dependent pathogenesis has yet to be clearly investigated. Recently, we identified an important role for tyrosine phosphatase Src homology domain-containing protein tyrosine phosphatase 2 (Shp2) in regulating the activation of alveolar macrophages. Over a long-term observation period, mice with Shp2 deletion in macrophages (LysMCre:Shp2fl/fl) develop spontaneous, progressive emphysema-like injury in the lungs, characterized by massive destruction of alveolar morphology, interstitial extracellular matrix degradation, and elevated levels of MMPs, particularly, significant increases of macrophage elastase (MMP12) in aged mice. Further analysis demonstrated that MMP12 suppression by TGF-β1 activation was apparently abrogated in LysMCre:Shp2fl/fl mice, whereas the TGF-β1 concentration in the lungs was relatively the same. Mechanistically, we found that loss of Shp2 resulted in attenuated SMAD2/3 phosphorylation and nuclear translocation in response to TGF-β activation, thereby upregulating MMP12 expression in macrophages. Together, our findings define a novel physiological function of Shp2 in TGF-β1/MMP12-dependent emphysema, adding insights into potential etiologies for this chronic lung disorder.

Pulmonary emphysema, one major manifestation of chronic obstructive pulmonary disease (COPD), is characterized by excessive destruction of the alveolar matrix, loss of lung elasticity, and permanent enlargement of distal airspaces, which ultimately leads to impaired oxygenation (1, 2). Emphysema is also a leading risk for morbidity and mortality of COPD patients worldwide, largely attributable to cigarette smoke (CS) exposure, for which therapeutic options are limited (3, 4). Previous studies have revealed that protease–antiprotease imbalance, oxidative stress, chronic lung inflammation, and alveolar cell apoptosis are critical steps involved in the pathogenesis of emphysema (58). Recently, alveolar macrophages (AMs) have been receiving emerging attention as important endogenous sources of matrix-degrading proteinases in the emphysematous lung from patients (9, 10). A variety of matrix metalloproteinases (MMPs) favoring destabilization, such as MMP-1, -2, -7, -9, and -12, are synthesized as proenzymes, which are then activated and secreted extracellularly by macrophages (11). The finding that broad-spectrum MMP inhibitors can prevent or effectively ameliorate emphysema in animal models further reinforces the importance of macrophages expressing MMPs. In vitro observations support the notion that activation of a number of MMPs correlates with the pathogenesis; nevertheless, the evidence is supportive for MMP12 as a therapeutic target in emphysema (12, 13). MMP12 is a 54-kDa elastolytic protease predominantly expressed by macrophages, and an increase in macrophage MMP12 has been observed clinically with the development of emphysema and COPD (1416). Mice lacking MMP12 have previously been reported to be protected from emphysema (17). TGF-β1 plays a pivotal role in regulation of the elastase–antielastase balance, contributing to extracellular matrix (ECM) homeostasis. Dysregulation of TGF-β1 signaling results in considerably increased levels of MMP12 and an age-related emphysematous phenotype in mouse models (1820). Notably, TGF-β1 concentration in serum is inversely correlated with emphysema severity (21). These studies suggest that functional alterations in TGF-β activation are closely related to MMP12-dependent emphysema; however, the mechanism underlying this process has yet to be clearly investigated, and in vivo evidence is also poorly described.

Src homology domain-containing protein tyrosine phosphatase (PTP) 2 (Shp2) is a cytoplasmic tyrosine phosphatase, containing two Src homology domains and one catalytic PTP domain (22, 23). Shp2 acts as a critical component of multiple signaling pathways involving a variety of physiological functions (2426). Shp2 is highly expressed in normal distal lungs and lung cancer (2729). Recent advances in targeted therapies with Shp2 inhibitors as anticancer therapeutic options have gained a great deal of attention (30). However, much remains to be learned about the in vivo function of Shp2 in the lungs. We previously reported that loss of Shp2 in alveoli epithelia led to pathological alteration of alveolar structures and lung mechanics, and eventually the development of spontaneous interstitial pulmonary fibrosis (31). Additionally, selective disruption of Shp2 in macrophages (LysMCre:Shp2fl/fl) skews macrophage activation toward M2 polarization. Although LysMCre:Shp2fl/fl mice appear to have normal lung histology at up to 6–8 wk of age, fibrotic lesions in LysMCre:Shp2fl/fl mice are severely aggravated in a model of bleomycin (BLM) toxicity (32).

Given that the activation of macrophages is altered because of Shp2 deficiency, we further investigated whether these Shp2 deficiency–related abnormalities cause long-term physiological outcomes in lungs. In this study, we reported that LysMCre:Shp2fl/fl mice developed progressive airspace enlargement and eventually exhibited spontaneous emphysematous lungs in aged mice. We further found that dysregulated TGF-β1 signaling resulting from the inactivation of Shp2, which disturbed ECM homeostasis, led to persistent elevation of MMP12 in lungs. These findings demonstrate the importance of Shp2 as a rheostat for MMP12-dependent emphysema through TGF-β1 signaling, adding fresh insight into the pathogenesis of emphysema.

Shp2flox/flox mice (26, 31) were mated with LysMCre/+ mice (33) to generate conditional Shp2 knockout mice. Shp2flox/flox mice were used as controls, and LysMCre/+:Shp2flox/flox were used as mutant mice (designated as Shp2fl/fl and LysMCre:Shp2fl/fl in this study, respectively) and were selected to be used in the experiments. To confirm the excision of Shp2, we used a forward (5′-CAGTTGCAACTTTCTTACCTC-3′) primer and a reverse (5′-GCAGGAGACTGCAGCTCAGTGATG-3′) primer within introns 3 and 4, respectively. All animal protocols were approved by the Animal Care and Use Committee of the Zhejiang University School of Medicine.

LysMCre:Shp2fl/fl and Shp2fl/fl mice were exposed to the smoke of 10 cigarettes using a smoking machine (TE-10; Teague Enterprises). The chambers were measured for total particulate matter concentrations of 160–180 mg/m3. The mice were exposed 2 h/d, 5 d/wk for up to 4 mo. The control group was exposed to filtered air.

Mice were euthanized by pentobarbital injection. Left lungs were tied at the bronchus and lavaged the right lung with 0.5 ml of Ca2+- and Mg2+-free PBS. The procedure was repeated three times to collect a total volume of 1.5 ml of bronchoalveolar lavage fluid (BALF). Then the collected BALF was cooled on ice and centrifuged at 1000 rpm for 10 min at 4°C. DMEM/F12 supplemented with 10% FBS with penicillin and streptomycin was used to resuspend the cell pellets. We then collected the adherent cells after 1 h and lysed them in radioimmunoprecipitation assay buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl (pH 8), and 0.1% SDS. The lysate was stored at −20°C until use.

Raw264.7 and A549 cells were cultured in RPMI 1640 and DMEM/High Glucose (HyClone, Logan, UT), respectively, supplemented with 10% FBS (HyClone), penicillin (100 U/ml), and streptomycin (100 mg/ml) (HyClone).

AMs were obtained from mice and gathered via quick adhesion. Peritoneal and bone marrow–derived macrophages (BMDMs) were isolated and differentiated as previously described (34, 35). Cells were cultured in DMEM/F12 supplemented with 10% FBS (HyClone), penicillin (100 U/ml), and streptomycin (100 mg/ml) (HyClone).

The lungs were digested and mechanically disintegrated to obtain single-cell suspensions, as previously described (36). Lung cell suspensions from LysMCre:Shp2fl/fl and Shp2fl/fl mice were stained with CD45-allophycocyanin, CD11b-PE, and F4/80-FITC. Flow cytometry analysis and cell sorting were performed on an AECA NovoCyte TM system (ACEA Biosciences, San Diego, CA) and a FACSAria II (BD Biosciences, Franklin Lakes, NJ), respectively. Data were analyzed with FlowJo software 7.6.

The plasmids were generated in the pXJ40 vector as previously described (37). The mutant plasmids were constructed with the Fast Mutagenesis System (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Plasmids were pXJ40-MYC-SHP2 (Shp2WT) and pXJ40-MYC-SHP2 gain-of-function mutants (Shp2D61G, Shp2A72G) or loss-of-function mutants (Shp2T468M, Shp2I282V).

Raw264.7 cells were cultured in 24-well plates (5 × 104 cells per well) overnight and then incubated with Shp2 shRNA lentiviral particles or control shRNA lentiviral particles, followed with lentiviral overexpression of vector, full-length Shp2 (Shp2WT), constitutively active Shp2 (Shp2A72G, Shp2D61G), or dominant-negative Shp2 (Shp2I282V) for up to 96 h.

BMDMs or Raw264.7 cells were first treated with or without TGF-β1 for the indicated times and washed by PBS. Cells (1 × 107) were harvested and kept at 4°C. Nuclear and cytoplasmic fractions were separated by a Nuclear and Cytoplasmic Extraction Kit (CWbiotech, Beijing, China).

Total RNA was extracted from lung tissues or cells using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNA was reversely transcribed into cDNA using the ReverTraAce qPCR (quantitative PCR) RT kit (Toyobo, Osaka, Japan). Real-time PCR was performed on a LightCycler Roche480 (Roche Molecular Systems) using the LightCycler Roche480 master kit. Results of real-time PCR were based on at least three independent experiments. All values were given as the ratios to β-actin levels. The sequences of the specific primers used were as follows: MMP12, 5′-GAGTCCAGCCACCAACATTAC-3′ and 5′-GCGAAGTGGGTCAAAGACAG-3′; MMP19, 5′-CTGTGGCTGGCATTCTTACTT-3′ and 5′-GGGCAGTCCAGATGCTTCC-3′; TIMP3, 5′-CTTCTGCAACTCCGACATCGT-3′ and 5′-GGGGCATCTTACTGAAGCCTC-3′; SMAD7, 5′-GGCCGGATCTCA-GGCATTC-3′ and 5′-TTGGGTATCTGGAGTAAGGAGG-3′; Smurf2, 5′-AAACAG-TTGCTTGGGAAGTCA-3′ and 5′-TGCTCAACACAGAAGGTATGGT-3′; SMAD4, 5′-GTCTGAGCATTGTGCATAGT-TTG-3′ and 5′-GACGGGCATAGA-TCACATGAG-3′.

AMs, BMDMs, and RAW264.7 cells were incubated in 24-well culture dishes at a density of 1 × 105 cells per well. Cells were washed by PBS and then fixed by 4% paraformaldehyde for 15 min and permeabilized using 0.1% Triton X-100 (Beyotime, Beijing, China) for 20 min. After blocking cells with 4% goat serum for 1 h, cells were cultured with primary Abs against SMAD2 and MMP12 overnight, both used at 1:100. Cells were treated with Alexa Fluor 594–conjugated secondary Ab (1:400; Invitrogen), and the nuclei were stained with DAPI. All the cells were imaged by an inverted confocal microscope (Carl Zeiss, Göttingen, Germany).

Radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl [pH 8], and 0.1% SDS) was used to treat proteins extracted from the cultured cells according to the protocols (Beyotime). After the measurement of protein concentration, a routine Western blot analysis was performed. Proteins were separated by SDS-PAGE on 10 or 13% polyacrylamide gels and then transferred to nitrocellulose membranes (Pall, Port Washington, NY). Five percent dry milk was diluted with TBST (150 mM NaCl, 50 mM Tris, 0.05% Tween 20 [pH 7.6]) to block unspecific sites for 1 h at room temperature. Subsequently, the membranes were incubated with primary Abs at 4°C overnight. Then the membranes were washed by TBST three times and treated with IRDye 680LT/IRDye 800CW secondary Abs (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature. Finally, the signals were detected by an Odyssey two-color infrared imaging system (LI-COR Biosciences).

Abs against Shp2 (Santa Cruz, Santa Cruz, CA); pSMAD2 (S465/467), pSMAD3 (S423/425), SMAD2, and SMAD3 (Cell Signaling Technologies, Beverly, MA); MMP12 (Abcam); β-actin, β-tubulin, Histone 3, and LMNB2 (HuaAn Biotechnology, Hangzhou, China); and IRDye 680LT/IRDye 800CW secondary Abs (LI-COR Biosciences) were used following the manufacturer’s instructions.

Lungs were fixed with an intratracheal injection of 4% paraformaldehyde in PBS at 25 cm H2O and immersed in the same fixative to maintain pulmonary architecture. Left lungs were embedded in paraffin. Then 5-μm-thick sections were cut and mounted on slides. H&E, Masson’s trichrome, and Verhoeff–Van Gieson staining were used to evaluate morphological changes, collagen deposition, and elastin fiber degradation in lungs, respectively. Assessment of distal airspace enlargement and alveolar wall destruction was performed as previously described (18, 38).

Data were given as mean ± SEM. A two-tailed Student t test was used to determine the mean differences. Statistical significance was considered when p < 0.05.

We previously introduced LysMCre:Shp2fl/fl mice in which the Shp2 gene was inactivated in macrophages. LysMCre:Shp2fl/fl mice develop normally and are apparently healthy up to 6–8 wk of age, but are susceptible to BLM toxicity (32). In this study, H&E staining of lung sections of LysMCre:Shp2fl/fl mice exhibits no visible abnormalities in alveolar morphology at 2 mo of age, but progressive emphysema-like observations at 8 and 15 mo of age (Fig. 1A). Pulmonary emphysema is pathologically characterized by destruction of the alveolar matrix, enlargement of distal airspaces, and loss of lung elasticity (39). As indicated in Fig. 1A–C, these changes caused by Shp2 deletion were characterized by widespread dilatation and distortion in alveolar architecture, which were further quantitatively assessed by measuring the mean linear intercept and destructive index of alveolar septa. Moreover, BALF macrophages from aged LysMCre:Shp2fl/fl mice appeared to be highly vacuolated with a marked swelling compared with that of control mice (Fig. 1D). These cellular morphological irregularities are clinically associated with the abnormal activation of macrophages in emphysematous human lungs. Lung sections from aged LysMCre:Shp2fl/fl mice displayed a decrease in the amount of the elastin fibers and collagen deposition in alveolar interstitium, as determined by Verhoeff–Van Gieson staining and Masson’s trichrome staining (Fig. 1E, 1F). Together, these findings suggest that mice with a selectively disrupted Shp2 in macrophages develop emphysema-like injury in an age-related manner.

FIGURE 1.

Shp2 deficiency leads to impaired alveolar architecture and emphysema-like injury. (A) Lung morphology of LysMCre:Shp2fl/fl and Shp2fl/fl mice. Lung sections from LysMCre:Shp2fl/fl and Shp2fl/fl mice (n = 6 per group) at 2, 8, and 15 mo of age were stained with H&E. (B) Quantitative analysis of histological lesions in alveolar space. Mean linear intercept (MLI) represents the alveolar enlargement (n = 6 per group). (C) Destructive index (DI) of lung sections from mice at indicated age (n = 6 per group). (D) Wright’s Giemsa staining of cytospin concentrated from BALF of LysMCre:Shp2fl/fl and Shp2fl/fl mice at 15 mo of age. Arrows indicate foamy, large, vacuolated AMs. (E) Elastin staining. Lung sections from LysMCre:Shp2fl/fl and Shp2fl/fl mice at 15 mo of age were Verhoeff–Van Gieson stained. Arrows indicate elastin in alveolar walls. (F) Measurement of collagen deposition by Masson’s trichrome staining of 15-mo-old lung sections. Scale bars, 100 μm (A, D, and F); 20 μm (E). Data from three independent experiments are shown. Error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 1.

Shp2 deficiency leads to impaired alveolar architecture and emphysema-like injury. (A) Lung morphology of LysMCre:Shp2fl/fl and Shp2fl/fl mice. Lung sections from LysMCre:Shp2fl/fl and Shp2fl/fl mice (n = 6 per group) at 2, 8, and 15 mo of age were stained with H&E. (B) Quantitative analysis of histological lesions in alveolar space. Mean linear intercept (MLI) represents the alveolar enlargement (n = 6 per group). (C) Destructive index (DI) of lung sections from mice at indicated age (n = 6 per group). (D) Wright’s Giemsa staining of cytospin concentrated from BALF of LysMCre:Shp2fl/fl and Shp2fl/fl mice at 15 mo of age. Arrows indicate foamy, large, vacuolated AMs. (E) Elastin staining. Lung sections from LysMCre:Shp2fl/fl and Shp2fl/fl mice at 15 mo of age were Verhoeff–Van Gieson stained. Arrows indicate elastin in alveolar walls. (F) Measurement of collagen deposition by Masson’s trichrome staining of 15-mo-old lung sections. Scale bars, 100 μm (A, D, and F); 20 μm (E). Data from three independent experiments are shown. Error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Pulmonary resident macrophages consist of two major subpopulations, namely AMs and interstitial macrophages (IMs) (40). To further assess the macrophage subpopulation with Shp2 deleted in our model, we performed flow cytometry analysis, and results showed that the numbers of CD45+F4/80+CD11b AMs were ∼10-fold higher than those in CD45+F4/80+CD11b+ IMs in 2-mo-old lungs, and the trend was similar in 15-mo-old lungs (Fig. 2A). AMs and IMs were further sorted from lung single-cell suspensions via FACS based on their differential CD45/F4/80/CD11b expression. RT-PCR analysis revealed that Shp2 mRNA expression was higher in AMs than in IMs (Fig. 2B), which was consistent with the Shp2 staining results determined by flow cytometry (Supplemental Fig. 1A). Furthermore, the efficiency of LysMCre-mediated genetic deletion of Shp2 was ∼70 and 50% in AMs and IMs, respectively (Fig. 2C). Considering the key roles of AMs in emphysema progression, together with our findings, we believe the importance of Shp2 in CD45+F4/80+CD11b AMs and their contribution to the phenotype observed in our study. In this work, conditional disruption of Shp2 in AMs was further confirmed (Fig. 2D, 2E, Supplemental Fig. 1B). Moreover, the numbers and ratio of AMs in LysMCre:Shp2fl/fl mice were similar to littermate controls (Supplemental Fig. 1C, 1D).

FIGURE 2.

Characterization of macrophage abundance and Shp2 expression levels in LysMCre:Shp2fl/fl mice. (A) Lung single-cell suspensions of LysMCre:Shp2fl/fl and Shp2fl/fl mice at 2 and 15 mo old were analyzed by flow cytometry. After the initial gating on viable CD45+ immune cells from lung single-cell suspensions, further characterization of the F4/80+ population revealed CD45+F4/80+CD11b AMs and CD45+F4/80+CD11b+ IMs. (B and C) Defined AMs and IMs from Shp2fl/fl and LysMCre:Shp2fl/fl mice at 2 mo old were further sorted via FACS. RT-PCR was used to measure Shp2 mRNA expression relative to β-actin. (D and E) Shp2 protein levels in purified AMs of LysMCre:Shp2fl/fl and Shp2fl/fl mice at 2 mo old were assessed by immunoblotting assays using Abs against Shp2. β-Actin was used as a loading control. Intensity of Shp2 relative to β-actin from each experiment was quantified by NIH Image software (n = 6 per group). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

Characterization of macrophage abundance and Shp2 expression levels in LysMCre:Shp2fl/fl mice. (A) Lung single-cell suspensions of LysMCre:Shp2fl/fl and Shp2fl/fl mice at 2 and 15 mo old were analyzed by flow cytometry. After the initial gating on viable CD45+ immune cells from lung single-cell suspensions, further characterization of the F4/80+ population revealed CD45+F4/80+CD11b AMs and CD45+F4/80+CD11b+ IMs. (B and C) Defined AMs and IMs from Shp2fl/fl and LysMCre:Shp2fl/fl mice at 2 mo old were further sorted via FACS. RT-PCR was used to measure Shp2 mRNA expression relative to β-actin. (D and E) Shp2 protein levels in purified AMs of LysMCre:Shp2fl/fl and Shp2fl/fl mice at 2 mo old were assessed by immunoblotting assays using Abs against Shp2. β-Actin was used as a loading control. Intensity of Shp2 relative to β-actin from each experiment was quantified by NIH Image software (n = 6 per group). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Overall, these results define a predominant function of Shp2 in AMs, rather than in IMs, in the regulation of emphysema-like injury in an age-related manner.

Pulmonary emphysema has been pathologically correlated with chronic inflammation and deregulated ECM homeostasis (41). BALF from adult and aged mice was assayed for cytokines, which indicated unaltered levels of IL-6, TNF-α, and IL-13 in LysMCre:Shp2fl/fl mice, compared with that of the controls at 2 and 15 mo (Supplemental Fig. 2A–C). To further assess the effect of Shp2 on ECM homeostasis, we investigated the mRNA levels of a variety of MMPs and TIMPs in AMs isolated from LysMCre:Shp2fl/fl and control mice. As indicated in Fig. 3A, we found a remarkable increase in the mRNA level of MMP12 and slightly elevated levels of MMP19 and TIMP3 in aged LysMCre:Shp2fl/fl mice at 15 mo. MMP12 is a macrophage-specific metalloelastase, which strongly degrades ECM components, and is sufficient for the development of emphysema (42). In this study, we focused on MMP12 expressed in AM, although IM was another source of MMP12 (Supplemental Fig. 1E). Elevation of MMP12 in AMs at the mRNA level was further confirmed and was consistent with the results of the protein level, as measured using immunoblotting and ELISA assays (Fig. 3B, 3C). In addition, we also observed considerably increased MMP12 immunostaining of AMs in cytospins (Fig. 3D). Lung tissues were further analyzed by immunoblotting and immunohistochemical staining, the results of which suggested an increase in MMP12 in LysMCre:Shp2fl/fl mice at 15 mo (Fig. 3E, 3F). Moreover, MMP12 is secreted as zymogen and becomes the active form after proteolytic cleavage; therefore, it would be important to measure MMP12 activity. As indicated in Fig. 3G, we revealed increased MMP12 activity in AMs from aged LysMCre:Shp2fl/fl mice. Collectively, these results indicate that loss of Shp2 in macrophages predisposes mice to pulmonary emphysema through alterations of macrophage MMP12 expression and activity.

FIGURE 3.

Increased MMP12 expression in lungs from aged LysMCre:Shp2fl/fl mice. (A) MMP mRNA expression profiles of AMs isolated by BALF from 2- to 15-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice. (B) Protein and mRNA expression levels of MMP12 in AMs from 2- to 8-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice. (C) Secreted MMP12 levels in BALF from 2- to 15-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice were determined by ELISA. (D) MMP12 immunostaining of AMs harvested from BALF. (E) Western blot analysis of MMP12 protein expression in lung tissues from LysMCre:Shp2fl/fl and Shp2fl/fl mice at indicated age. (F) Immunohistochemical analysis of MMP12 in lung sections. (G) MMP12 activity in BALF was detected by Sensolyte MMP12 assay kit. Data from three independent experiments are shown. Scale bar, 10 μm (D); 20 μm (F). Error bars indicate mean ± SEM. *p < 0.05, **p < 0.01.

FIGURE 3.

Increased MMP12 expression in lungs from aged LysMCre:Shp2fl/fl mice. (A) MMP mRNA expression profiles of AMs isolated by BALF from 2- to 15-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice. (B) Protein and mRNA expression levels of MMP12 in AMs from 2- to 8-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice. (C) Secreted MMP12 levels in BALF from 2- to 15-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice were determined by ELISA. (D) MMP12 immunostaining of AMs harvested from BALF. (E) Western blot analysis of MMP12 protein expression in lung tissues from LysMCre:Shp2fl/fl and Shp2fl/fl mice at indicated age. (F) Immunohistochemical analysis of MMP12 in lung sections. (G) MMP12 activity in BALF was detected by Sensolyte MMP12 assay kit. Data from three independent experiments are shown. Scale bar, 10 μm (D); 20 μm (F). Error bars indicate mean ± SEM. *p < 0.05, **p < 0.01.

Close modal

TGF-β1 is known to play a pivotal role in age-dependent pulmonary emphysema by downregulating MMP12 expression (18, 43, 44). To examine this possibility, we first examined TGF-β1 levels of BALF from LysMCre:Shp2fl/fl and control mice at 2 and 15 mo, suggesting that Shp2 deficiency had no effects on TGF-β1 production in vivo (Fig. 4A). Then AMs and peritoneal macrophages (PMs) isolated from LysMCre:Shp2fl/fl and control mice were treated with TGF-β1 for MMP12 measurement. As illustrated in Fig. 4B, 4C, and 4E, MMP12 expression was suppressed in control mice after TGF-β1 treatment, whereas it was evident that the effect of MMP12 suppression was insufficient in LysMCre:Shp2fl/fl mice in response to TGF-β1 at both mRNA and protein levels. Consistently, when macrophage cell line Raw264.7 was pharmacologically treated with Shp2 inhibitor (phenylhydrazonopyrazolone sulfonate 1 [PHPS1]) in vitro, the inhibition effect of TGF-β1 on MMP12 expression was downregulated (Fig. 4D). Together, these findings suggest that the emphysematous lungs observed in aged LysMCre:Shp2fl/fl mice are attributable to dysregulation of TGF-β1–mediated MMP12 suppression.

FIGURE 4.

Ablation of Shp2 blocks TGF-β1–mediated MMP12 suppression. (A) TGF-β1 expression levels in BALF from 2- to 15-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice were determined by ELISA. (B and C) AMs and PMs from 2-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice with or without TGF-β1 treatment (10 ng/ml) for 24 h. (D) Raw264.7 cells were pretreated with Shp2 inhibitor PHPS1 (20 μM) or the equivalent volume of DMSO for 2 h and then stimulated with TGF-β1 (10 ng/ml) for 24 h. (B–D) RT-PCR was used to measure the transcript levels of MMP12 relative to β-actin. (E) PMs from 2-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice incubated with TGF-β1 (10 ng/ml) for 24 and 48 h. MMP12 protein levels were assessed by Western blot analysis. Data from three independent experiments are shown. Error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

Ablation of Shp2 blocks TGF-β1–mediated MMP12 suppression. (A) TGF-β1 expression levels in BALF from 2- to 15-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice were determined by ELISA. (B and C) AMs and PMs from 2-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice with or without TGF-β1 treatment (10 ng/ml) for 24 h. (D) Raw264.7 cells were pretreated with Shp2 inhibitor PHPS1 (20 μM) or the equivalent volume of DMSO for 2 h and then stimulated with TGF-β1 (10 ng/ml) for 24 h. (B–D) RT-PCR was used to measure the transcript levels of MMP12 relative to β-actin. (E) PMs from 2-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice incubated with TGF-β1 (10 ng/ml) for 24 and 48 h. MMP12 protein levels were assessed by Western blot analysis. Data from three independent experiments are shown. Error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

TGF-β1 signaling is initiated by TGF-βR activation, thereby targeting cytoplasmic SMADs (45). To investigate the potential mechanism by which Shp2 is involved in TGF-β1 activation, leading to MMP12 suppression, we first assessed the expression of TGF-β receptors I and II and two major SMAD components (SMAD2/3) in macrophages in vitro. As shown in Fig. 5A, TGF-β1–induced SMAD2/3 activation was diminished in Raw264.7 cells pretreated with PHPS1 in time-course experiments. Consistently, impaired in vivo activation of SMAD2/3 was observed in AMs, PMs and BMDMs, in response to TGF-β1 stimulation (Fig. 5B–D), whereas the expression of two major negative components (SMAD7 and Smurf2) and co-SMAD (SMAD4) remained unaltered, which is known to be indicative of active TGF-β1 signaling (Supplemental Fig. 3A–E). Collectively, these evidences indicate that Shp2 positively regulates TGF-β1 signaling mainly through SMAD2/3 activation.

FIGURE 5.

Loss of Shp2 inhibits TGF-β1–mediated SMAD2/3 activation. (A) Raw264.7 cells were pretreated with Shp2 inhibitor PHPS1 (20 μM) or DMSO for 2 h, followed by TGF-β1 (10 ng/ml) stimulation for 0, 5, 30, 60, 120, or 360 min. TGF-β receptor II (TβRII), p-SMAD2, SMAD2, p-SMAD3, and SMAD3 were analyzed by Western blot analysis. (B) AMs isolated from 2-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice were treated with TGF-β1 (10 ng/ml) for 0 or 30 min. Total cell lysates were used to quantify levels of Shp2, total SMAD2/3, and p-SMAD2/3. (C and D) PMs and BMDMs from 2-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice were treated with TGF-β1 (10 ng/ml) for the indicated time periods. Cells were collected and lysed for immunoblotting analysis after stimulation. Data from three independent experiments are shown.

FIGURE 5.

Loss of Shp2 inhibits TGF-β1–mediated SMAD2/3 activation. (A) Raw264.7 cells were pretreated with Shp2 inhibitor PHPS1 (20 μM) or DMSO for 2 h, followed by TGF-β1 (10 ng/ml) stimulation for 0, 5, 30, 60, 120, or 360 min. TGF-β receptor II (TβRII), p-SMAD2, SMAD2, p-SMAD3, and SMAD3 were analyzed by Western blot analysis. (B) AMs isolated from 2-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice were treated with TGF-β1 (10 ng/ml) for 0 or 30 min. Total cell lysates were used to quantify levels of Shp2, total SMAD2/3, and p-SMAD2/3. (C and D) PMs and BMDMs from 2-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice were treated with TGF-β1 (10 ng/ml) for the indicated time periods. Cells were collected and lysed for immunoblotting analysis after stimulation. Data from three independent experiments are shown.

Close modal

It has been well demonstrated that upon TGF-β1 activation, cytoplasmic SMAD2/3 are phosphorylated and translocated into the nucleus, where SMAD3 serve as transcriptional repressors, directly binding with promoter regions of MMP12 (46). To further evaluate the functional relevance of Shp2 interference with MMP12 expression, we first examined whether inactivation of Shp2 causes altered activation of nuclear SMADs. Nuclear and cytoplasmic fractions obtained from Raw264.7 cells after TGF-β1 stimulation were analyzed with immunoblotting assays. As shown in Fig. 6A and 6B, levels of phosphorylated SMAD2 were reduced in the nucleus of PHPS1-pretreated macrophages in vitro in a time- and dose-dependent manner. Furthermore, in vivo BMDMs were further purified and stimulated with TGF-β1 for indicated time periods, which was followed by immunoblotting analysis with cytosolic-nuclear fragments. Consistently, these results showed that inactivation of Shp2 evidently attenuated the nuclear aggregation of phosphorylated SMAD2 (Fig. 6C). Next, the reduction in nuclear translocation of SMAD2 after Shp2 inactivation was visualized using an immunostaining assay. As described in Fig. 6D, we observed that the nuclear staining of SMAD2 was reduced in LysMCre:Shp2fl/fl BMDMs in response to TGF-β1 compared with that of the controls. Similarly, immunofluorescence revealed that PHPS1 significantly attenuated nuclear translocation of SMAD2 in response to TGF-β1 (Supplemental Fig. 2D). Moreover, in the absence of Shp2 by using the lentivirus-mediated system, lentiviral overexpression of constitutively active Shp2 (Shp2D61G and Shp2A72G) promoted the phosphorylation of SMAD2, whereas dominant-negative Shp2 (Shp2I282V) impaired SMAD2 activation in TGF-β1 stimulation (Fig. 6E). Using these loss-of-function and gain-of-function mutants of Shp2, we performed a CAGA-luciferase reporter activity assay, which further confirmed that upregulation of Shp2 was required for TGF-β1–responsive elements, in a catalytically dependent (Shp2-PTP domain) manner (Fig. 6F). Hence, we suppose that Shp2 deficiency impairs TGF-β1–mediated SMAD2/3 activation, which results in the upregulation of the TGF-β1 target gene MMP12, eventually leading to elastin degradation and emphysematous lung in aged mice (Supplemental Fig. 3F).

FIGURE 6.

Shp2 regulates phosphorylation and nuclear export of SMAD2 in a PTP-dependent manner. (A) Raw264.7 cells were pretreated with PHPS1 (20 μM) or DMSO for 2 h and then incubated with TGF-β1 (10 ng/ml) for 0, 5, 30, 60, or 120 min. Cytoplasmic and nuclear fractions were subjected to immunoblotting. β-Tubulin and LMNB2 were used as loading controls. (B) Raw264.7 cells were pretreated with PHPS1 (0, 0.2, 2, 20, or 100 μM), followed by TGF-β1 (10 ng/ml) stimulation. Cytoplasmic and nuclear fractions were subjected to immunoblotting. β-Tubulin and LMNB2 were used as loading controls. (C) BMDMs from 2-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice were treated with TGF-β1 (10 ng/ml) for the indicated time periods. Cytoplasmic and nuclear fractions were subjected to immunoblotting. β-Actin and Histone 3 were used as loading controls. (D) Adherent BMDMs were treated with TGF-β1 (10 ng/ml) for 0 or 120 min for confocal imaging analysis. SMAD2 was visualized using anti-SMAD2 Ab (FITC). DAPI (DNA staining) and merged images are indicated. Scale bar, 20 μm. (E) Raw264.7 cells were first transduced with lentiviruses encoding control shRNA or Shp2 shRNA, followed with overexpression of vector, full-length Shp2 (Shp2WT), constitutively active Shp2 (Shp2A72G, Shp2D61G), or dominant-negative Shp2 (Shp2I282V) via lentivirus infection, respectively. Cells were followed by TGF-β1 (10 ng/ml) stimulation and lysed for immunoblotting. (F) A549 cells transfected with CAGA-luciferase reporter and indicated Shp2 mutants were treated with TGF-β1 (10 ng/ml) for 18 h and then harvested for luciferase assay. Data from three independent experiments are shown. *p < 0.05, ***p < 0.001.

FIGURE 6.

Shp2 regulates phosphorylation and nuclear export of SMAD2 in a PTP-dependent manner. (A) Raw264.7 cells were pretreated with PHPS1 (20 μM) or DMSO for 2 h and then incubated with TGF-β1 (10 ng/ml) for 0, 5, 30, 60, or 120 min. Cytoplasmic and nuclear fractions were subjected to immunoblotting. β-Tubulin and LMNB2 were used as loading controls. (B) Raw264.7 cells were pretreated with PHPS1 (0, 0.2, 2, 20, or 100 μM), followed by TGF-β1 (10 ng/ml) stimulation. Cytoplasmic and nuclear fractions were subjected to immunoblotting. β-Tubulin and LMNB2 were used as loading controls. (C) BMDMs from 2-mo-old LysMCre:Shp2fl/fl and Shp2fl/fl mice were treated with TGF-β1 (10 ng/ml) for the indicated time periods. Cytoplasmic and nuclear fractions were subjected to immunoblotting. β-Actin and Histone 3 were used as loading controls. (D) Adherent BMDMs were treated with TGF-β1 (10 ng/ml) for 0 or 120 min for confocal imaging analysis. SMAD2 was visualized using anti-SMAD2 Ab (FITC). DAPI (DNA staining) and merged images are indicated. Scale bar, 20 μm. (E) Raw264.7 cells were first transduced with lentiviruses encoding control shRNA or Shp2 shRNA, followed with overexpression of vector, full-length Shp2 (Shp2WT), constitutively active Shp2 (Shp2A72G, Shp2D61G), or dominant-negative Shp2 (Shp2I282V) via lentivirus infection, respectively. Cells were followed by TGF-β1 (10 ng/ml) stimulation and lysed for immunoblotting. (F) A549 cells transfected with CAGA-luciferase reporter and indicated Shp2 mutants were treated with TGF-β1 (10 ng/ml) for 18 h and then harvested for luciferase assay. Data from three independent experiments are shown. *p < 0.05, ***p < 0.001.

Close modal

Emphysema is a progressive, incurable lung condition with high morbidity and mortality; alterations evidently are marked by alveolar destruction and enlargement with irreversible airflow limitation (47). Activated macrophages in emphysematous lungs are known to regulate homeostasis of the ECM, which is a crucial pathogenetic event for this disease. Previous studies have demonstrated an important role for TGF-β in regulating macrophage elastase MMP12 activity in the development of emphysema. However, the understanding of the mechanisms responsible for this cellular process has not been fully elucidated.

In our study, we introduced LysMCre:Shp2fl/fl mice in which Shp2 is disrupted in myeloid cells (including macrophages, neutrophils, and eosinophils) and other cell types (such as lymphocytes and neurons) as previously described (48, 49). Considering the undetectable inflammation (Supplemental Fig. 2A–C) and the contribution of Shp2 in AMs in our mouse model (Fig. 2), we focused our study on AMs without addressing IMs, neutrophils, and other cell types. In LysMCre:Shp2fl/fl mice, the functional inhibition of Shp2 in other cell types cannot be ignored, and future investigations should evaluate these further, along with the specific roles of the various cell types.

In this work, we first provided genetic evidence that mice carrying macrophage deletion of Shp2 (LysMCre:Shp2fl/fl) developed age-related spontaneous emphysema-like alterations (Fig. 1). In support, we found the most notable level of MMP12 elevation in LysMCre:Shp2fl/fl mice, possibly contributing to the observed phenotypic defects (Fig. 3). Shp2 deficiency relieved TGF-β1–mediated MMP12 suppression (Fig. 4). Furthermore, Shp2 positively regulated SMAD2/3 phosphorylation and nuclear translocation in response to TGF-β1 (Fig. 5), and overexpression of constitutively active or the dominant-negative of Shp2 mutants significantly affected TGF-β1–mediated MMP12 suppression (Fig. 6). These findings defined a novel role of Shp2 in the regulation of MMPs by TGF-β1, adding insight into the pathogenesis of pulmonary emphysema.

Shp2 is a ubiquitously expressed nonreceptor PTP with two tandem N-terminal Src homology domains and one C-terminal catalytic PTP domain. Emerging evidence has revealed a broad spectrum of cellular functions regulated by Shp2, acting downstream of various growth factors and cytokine receptors (5052). Homozygous Shp2-mutant mice exhibit early embryonic lethality at midgestation (embryonic days 8.5–10.5) with multiple organogenetic defects (53). Homozygous Shp2-mutant mice carrying a mutant allele of EGFR (wa-2) appear more frequently in postnatal morbidity (postnatal day 10.5). Histological analysis reveals severe morphological defects in distal lungs, as evidenced by a marked reduction in the size of alveolar spaces and an increase in the thickness of alveolar walls (54). This impaired alveolarization suggests a putative requirement of Shp2 for adult pulmonary physiology, which prompts us to explore further the precise roles of Shp2 in lungs. Given that Shp2 is a widely expressed phosphatase, its signaling is temporally and spatially regulated, and thus the Cre-loxP recombinase system holds great promise as a genetic strategy, enabling the precise excision of a targeted gene in a specific tissue (55, 56). Using a combined Cre-loxP–mediated inducible knockout approach, we previously introduced a triple-transgenic Shp2-knockout [SP-C-rtTA/(tetO)7-Cre/Shp2flox/flox], of which mice at 4–6 wk old were fed with doxycycline to transiently abolish Shp2 in alveoli, leading to dysregulated surfactant hemostasis, disorganized alveolar architecture, and fibrotic remodeling of the lungs (31). In addition, we further engineered LysMCre:Shp2fl/fl mice, which showed that macrophages lacking Shp2 were skewed toward an alternatively activated polarization and aggravated fibrotic injury in challenge of the BLM model (32). This genetic evidence supports the functional requirement of Shp2 in alveolar integrity and physiology; however, long-term physiological outcomes for Shp2 deficiency in the lungs remain unclear. In this article, we reported the function of Shp2 in long-term alveolar physiology, and the results provide direct evidence for a conserved requirement of Shp2 in TGF-β1–mediated ECM homeostasis in the prevention of emphysema. Notably, pharmacological targeting of Shp2 in lung cancer recently has been receiving considerable attention as a novel promising therapeutic option (30). Nevertheless, it raises questions whether this inactivation could result in potential toxicities in lung. In this study, our phenotypic observation predicted by mouse genetic analysis has led to a more comprehensive understanding into this issue.

ECM homeostasis is thought to play an important role in the pathogenesis of emphysema (57). Furthermore, of all the MMPs, macrophage elastase (MMP12) is a key entity in this process. Clinical study has revealed that MMP12 activity is markedly enhanced in sputum from COPD patients (14). In addition, several genetic models, such as surfactant-associated protein D or TLR4 deficiency in mice, exhibit increased elastolytic activity, resulting in MMP12-related emphysema (58, 59). Notably, MMP12−/− knockout mice do not develop emphysema after long-term exposure to CS (17). These studies emphasize a central role of MMP12 in emphysematous destruction. In this study, we searched for candidate genes associated with the pathogenesis of emphysema in LysMCre:Shp2fl/fl mice. We identified MMP12 as a predominately highly upregulated gene in aged LysMCre:Shp2fl/fl macrophages, whereas the expression of other MMPs/TIMPs remained almost unchanged. Taken together, our results in mice support the relationship between MMP12 and the pathogenesis of pulmonary emphysema studied previously.

MMP12 expression is upregulated by a variety of cytokines and growth factors, for example, by IFN-γ, IL-17, and epidermal growth factor (6062). Given this variety, MMP12 inhibition appears to be more feasible than any attempts to selectively prevent its production. Notably, TGF-β1 signaling is intimately tied to MMP12 expression. Moreover, TGF-β1 plays a pivotal role in regulating matrix homeostasis and global lung tissue remodeling. Indeed, mice with integrin αvβ6 or SMAD3 deficiency display diminished TGF-β1 signaling, which causes MMP12-dependent spontaneous age-dependent emphysema (18, 19). Although alternations of TGF-β1 signaling have been studied extensively, the correlation between the TGF-β1 pathway and tyrosine phosphatase Shp2 in the process of emphysema-like injury has yet to be studied. Our results suggest a novel role for Shp2 in modulating TGF-β1 signaling and TGF-β1 target genes in emphysematous conditions. We show that Shp2 positively regulates the phosphorylation and nuclear export of SMAD2/3, whereas other key proteins are unaltered. Moreover, PTP enzyme activity is required in this process. SMAD3 is essential for transcriptional repression of the MMP12 promoter by TGF-β1 (46). Shp2 deficiency results in a reduction in the phosphorylation level and the nuclear accumulation of SMAD2/3, which leads to MMP12 overexpression, thereby contributing to emphysema.

Chronic exposure to CS is a pivotal risk factor for lung injury, which contributes to the development of emphysema. Previous study has shown that prominent reductions in TGF-β signaling and decreased TIMP3 were found in a smoke-induced lung injury model, which augmented emphysema in these mice. In our work, we have reported a new role for Shp2 to act as a positive regulator of TGF-β/MMP12 signaling in spontaneous emphysematous lungs. To further support this molecular explanation in lung injury, we also employed a CS-induced lung injury model. Our data revealed that CS exposure significantly increased the number of total leukocytes in BALF, especially macrophages, but no statistical difference was found between LysMCre:Shp2fl/fl and Shp2fl/fl mice (Supplemental Fig. 4A). In addition, the production of inflammatory cytokines IL-6 and TNF-α in BALF remained unchanged in Shp2-deficient mice and the controls (Supplemental Fig. 4B). LysMCre:Shp2fl/fl mice were more susceptible to CS-induced lung injury featuring more severe airspace enlargement, accompanied by an elevation of MMP12 in AMs, in response to chronic CS exposure (Supplemental Fig. 4C–E). Collectively, these results further proved the important role of Shp2 in regulating MMP12-mediated lung injury.

In summary, our findings provide a novel molecular mechanism of emphysema pathogenesis by linking Shp2 with TGF-β1/MMP12 signaling, which may serve as a new genetic animal model for emphysema-like lung injury and provide a therapeutic option for this currently untreatable lung disease.

We thank G.S. Feng (University of California, San Diego, San Diego, CA) for providing the Shp2flox/flox mice, the facility of Microscopic Imaging, Zhejiang University School of Medicine for assistance with confocal microscopy, and W. Ning (College of Life Sciences, Nankai University) for providing the CAGA-luciferase-reporter plasmid.

This work was supported by National Natural Science Foundation of China Grants 81530001 (to Y.K.), 31471258 (to H.C.), and 31370857 (to X.Z.) and by Zhejiang Provincial Natural Science Foundation of China Grant LY15H040009 (to Z.Z.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • AM

    alveolar macrophage

  •  
  • BALF

    bronchoalveolar lavage fluid

  •  
  • BLM

    bleomycin

  •  
  • BMDM

    bone marrow–derived macrophage

  •  
  • COPD

    chronic obstructive pulmonary disease

  •  
  • CS

    cigarette smoke

  •  
  • ECM

    extracellular matrix

  •  
  • IM

    interstitial macrophage

  •  
  • MMP

    matrix metalloproteinase

  •  
  • PHPS1

    phenylhydrazonopyrazolone sulfonate 1

  •  
  • PM

    peritoneal macrophage

  •  
  • PTP

    protein tyrosine phosphatase

  •  
  • Shp2

    Src homology domain-containing PTP 2.

1
Snider
,
G. L.
,
J. L.
Kleinerman
,
W. M.
Thurlbeck
,
Z. H.
Bengali
.
1985
.
The definition of emphysema. Report of a National Heart, Lung, and Blood Institute, Division of Lung Diseases workshop.
Am. Rev. Respir. Dis.
132
:
182
185
.
2
Petrache
,
I.
,
V.
Natarajan
,
L.
Zhen
,
T. R.
Medler
,
A. T.
Richter
,
C.
Cho
,
W. C.
Hubbard
,
E. V.
Berdyshev
,
R. M.
Tuder
.
2005
.
Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice.
Nat. Med.
11
:
491
498
.
3
Murray
,
C. J.
,
A. D.
Lopez
.
1997
.
Alternative projections of mortality and disability by cause 1990-2020: global burden of disease study.
Lancet
349
:
1498
1504
.
4
Seimetz
,
M.
,
N.
Parajuli
,
A.
Pichl
,
F.
Veit
,
G.
Kwapiszewska
,
F. C.
Weisel
,
K.
Milger
,
B.
Egemnazarov
,
A.
Turowska
,
B.
Fuchs
, et al
.
2011
.
Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice.
Cell
147
:
293
305
.
5
Elkington
,
P. T.
,
J. S.
Friedland
.
2006
.
Matrix metalloproteinases in destructive pulmonary pathology.
Thorax
61
:
259
266
.
6
Greenlee
,
K. J.
,
Z.
Werb
,
F.
Kheradmand
.
2007
.
Matrix metalloproteinases in lung: multiple, multifarious, and multifaceted.
Physiol. Rev.
87
:
69
98
.
7
Kasahara
,
Y.
,
R. M.
Tuder
,
L.
Taraseviciene-Stewart
,
T. D.
Le Cras
,
S.
Abman
,
P. K.
Hirth
,
J.
Waltenberger
,
N. F.
Voelkel
.
2000
.
Inhibition of VEGF receptors causes lung cell apoptosis and emphysema.
J. Clin. Invest.
106
:
1311
1319
.
8
Tuder
,
R. M.
,
I.
Petrache
.
2012
.
Pathogenesis of chronic obstructive pulmonary disease.
J. Clin. Invest.
122
:
2749
2755
.
9
Shapiro
,
S. D.
1994
.
Elastolytic metalloproteinases produced by human mononuclear phagocytes. Potential roles in destructive lung disease.
Am. J. Respir. Crit. Care Med.
150
:
S160
S164
.
10
Ishii
,
T.
,
R. T.
Abboud
,
A. M.
Wallace
,
J. C.
English
,
H. O.
Coxson
,
R. J.
Finley
,
K.
Shumansky
,
P. D.
Paré
,
A. J.
Sandford
.
2014
.
Alveolar macrophage proteinase/antiproteinase expression in lung function and emphysema.
Eur. Respir. J.
43
:
82
91
.
11
Houghton
,
A. M.
2015
.
Matrix metalloproteinases in destructive lung disease.
Matrix Biol.
44–46
:
167
174
.
12
Churg
,
A.
,
M.
Cosio
,
J. L.
Wright
.
2008
.
Mechanisms of cigarette smoke-induced COPD: insights from animal models.
Am. J. Physiol. Lung Cell. Mol. Physiol.
294
:
L612
L631
.
13
Vandenbroucke
,
R. E.
,
E.
Dejonckheere
,
C.
Libert
.
2011
.
A therapeutic role for matrix metalloproteinase inhibitors in lung diseases?
Eur. Respir. J.
38
:
1200
1214
.
14
Demedts
,
I. K.
,
A.
Morel-Montero
,
S.
Lebecque
,
Y.
Pacheco
,
D.
Cataldo
,
G. F.
Joos
,
R. A.
Pauwels
,
G. G.
Brusselle
.
2006
.
Elevated MMP-12 protein levels in induced sputum from patients with COPD.
Thorax
61
:
196
201
.
15
Molet
,
S.
,
C.
Belleguic
,
H.
Lena
,
N.
Germain
,
C. P.
Bertrand
,
S. D.
Shapiro
,
J. M.
Planquois
,
P.
Delaval
,
V.
Lagente
.
2005
.
Increase in macrophage elastase (MMP-12) in lungs from patients with chronic obstructive pulmonary disease.
Inflammation Res.
54
:
31
36
.
16
Hunninghake
,
G. M.
,
M. H.
Cho
,
Y.
Tesfaigzi
,
M. E.
Soto-Quiros
,
L.
Avila
,
J.
Lasky-Su
,
C.
Stidley
,
E.
Melén
,
C.
Söderhäll
,
J.
Hallberg
, et al
.
2009
.
MMP12, lung function, and COPD in high-risk populations.
N. Engl. J. Med.
361
:
2599
2608
.
17
Hautamaki
,
R. D.
,
D. K.
Kobayashi
,
R. M.
Senior
,
S. D.
Shapiro
.
1997
.
Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice.
Science
277
:
2002
2004
.
18
Morris
,
D. G.
,
X.
Huang
,
N.
Kaminski
,
Y.
Wang
,
S. D.
Shapiro
,
G.
Dolganov
,
A.
Glick
,
D.
Sheppard
.
2003
.
Loss of integrin alpha(v)beta6-mediated TGF-beta activation causes Mmp12-dependent emphysema.
Nature
422
:
169
173
.
19
Bonniaud
,
P.
,
M.
Kolb
,
T.
Galt
,
J.
Robertson
,
C.
Robbins
,
M.
Stampfli
,
C.
Lavery
,
P. J.
Margetts
,
A. B.
Roberts
,
J.
Gauldie
.
2004
.
Smad3 null mice develop airspace enlargement and are resistant to TGF-beta-mediated pulmonary fibrosis.
J. Immunol.
173
:
2099
2108
.
20
Wang
,
X.
,
S.
Inoue
,
J.
Gu
,
E.
Miyoshi
,
K.
Noda
,
W.
Li
,
Y.
Mizuno-Horikawa
,
M.
Nakano
,
M.
Asahi
,
M.
Takahashi
, et al
.
2005
.
Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.
Proc. Natl. Acad. Sci. USA
102
:
15791
15796
.
21
Kamio
,
K.
,
T.
Ishii
,
T.
Motegi
,
K.
Hattori
,
Y.
Kusunoki
,
A.
Azuma
,
A.
Gemma
,
K.
Kida
.
2013
.
Decreased serum transforming growth factor-β1 concentration with aging is associated with the severity of emphysema in chronic obstructive pulmonary disease.
Geriatr. Gerontol. Int.
13
:
1069
1075
.
22
Feng
,
G. S.
,
C. C.
Hui
,
T.
Pawson
.
1993
.
SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases.
Science
259
:
1607
1611
.
23
Feng
,
G. S.
1999
.
Shp-2 tyrosine phosphatase: signaling one cell or many.
Exp. Cell Res.
253
:
47
54
.
24
Bard-Chapeau
,
E. A.
,
S.
Li
,
J.
Ding
,
S. S.
Zhang
,
H. H.
Zhu
,
F.
Princen
,
D. D.
Fang
,
T.
Han
,
B.
Bailly-Maitre
,
V.
Poli
, et al
.
2011
.
Ptpn11/Shp2 acts as a tumor suppressor in hepatocellular carcinogenesis.
Cancer Cell
19
:
629
639
.
25
Zhu
,
H. H.
,
K.
Ji
,
N.
Alderson
,
Z.
He
,
S.
Li
,
W.
Liu
,
D. E.
Zhang
,
L.
Li
,
G. S.
Feng
.
2011
.
Kit-Shp2-Kit signaling acts to maintain a functional hematopoietic stem and progenitor cell pool.
Blood
117
:
5350
5361
.
26
Ke
,
Y.
,
E. E.
Zhang
,
K.
Hagihara
,
D.
Wu
,
Y.
Pang
,
R.
Klein
,
T.
Curran
,
B.
Ranscht
,
G. S.
Feng
.
2007
.
Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality.
Mol. Cell. Biol.
27
:
6706
6717
.
27
Tefft
,
D.
,
S. P.
De Langhe
,
P. M.
Del Moral
,
F.
Sala
,
W.
Shi
,
S.
Bellusci
,
D.
Warburton
.
2005
.
A novel function for the protein tyrosine phosphatase Shp2 during lung branching morphogenesis.
Dev. Biol.
282
:
422
431
.
28
Bentires-Alj
,
M.
,
J. G.
Paez
,
F. S.
David
,
H.
Keilhack
,
B.
Halmos
,
K.
Naoki
,
J. M.
Maris
,
A.
Richardson
,
A.
Bardelli
,
D. J.
Sugarbaker
, et al
.
2004
.
Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia.
Cancer Res.
64
:
8816
8820
.
29
Chan
,
G.
,
D.
Kalaitzidis
,
B. G.
Neel
.
2008
.
The tyrosine phosphatase Shp2 (PTPN11) in cancer.
Cancer Metastasis Rev.
27
:
179
192
.
30
Chen
,
Y. N.
,
M. J.
LaMarche
,
H. M.
Chan
,
P.
Fekkes
,
J.
Garcia-Fortanet
,
M. G.
Acker
,
B.
Antonakos
,
C. H.
Chen
,
Z.
Chen
,
V. G.
Cooke
, et al
.
2016
.
Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.
Nature
535
:
148
152
.
31
Zhang
,
X.
,
Y.
Zhang
,
B.
Tao
,
L.
Teng
,
Y.
Li
,
R.
Cao
,
Q.
Gui
,
M.
Ye
,
X.
Mou
,
H.
Cheng
, et al
.
2012
.
Loss of Shp2 in alveoli epithelia induces deregulated surfactant homeostasis, resulting in spontaneous pulmonary fibrosis.
FASEB J.
26
:
2338
2350
.
32
Tao
,
B.
,
W.
Jin
,
J.
Xu
,
Z.
Liang
,
J.
Yao
,
Y.
Zhang
,
K.
Wang
,
H.
Cheng
,
X.
Zhang
,
Y.
Ke
.
2014
.
Myeloid-specific disruption of tyrosine phosphatase Shp2 promotes alternative activation of macrophages and predisposes mice to pulmonary fibrosis.
J. Immunol.
193
:
2801
2811
.
33
Clausen
,
B. E.
,
C.
Burkhardt
,
W.
Reith
,
R.
Renkawitz
,
I.
Förster
.
1999
.
Conditional gene targeting in macrophages and granulocytes using LysMcre mice.
Transgenic Res.
8
:
265
277
.
34
Herbert
,
D. R.
,
C.
Hölscher
,
M.
Mohrs
,
B.
Arendse
,
A.
Schwegmann
,
M.
Radwanska
,
M.
Leeto
,
R.
Kirsch
,
P.
Hall
,
H.
Mossmann
, et al
.
2004
.
Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology.
Immunity
20
:
623
635
.
35
Keul
,
P.
,
S.
Lucke
,
K.
von Wnuck Lipinski
,
C.
Bode
,
M.
Gräler
,
G.
Heusch
,
B.
Levkau
.
2011
.
Sphingosine-1-phosphate receptor 3 promotes recruitment of monocyte/macrophages in inflammation and atherosclerosis.
Circ. Res.
108
:
314
323
.
36
Sharma
,
S. K.
,
N. K.
Chintala
,
S. K.
Vadrevu
,
J.
Patel
,
M.
Karbowniczek
,
M. M.
Markiewski
.
2015
.
Pulmonary alveolar macrophages contribute to the premetastatic niche by suppressing antitumor T cell responses in the lungs.
J. Immunol.
194
:
5529
5538
.
37
Cheng
,
H.
,
K.
Kimura
,
A. K.
Peter
,
L.
Cui
,
K.
Ouyang
,
T.
Shen
,
Y.
Liu
,
Y.
Gu
,
N. D.
Dalton
,
S. M.
Evans
, et al
.
2010
.
Loss of enigma homolog protein results in dilated cardiomyopathy.
Circ. Res.
107
:
348
356
.
38
Saetta
,
M.
,
R. J.
Shiner
,
G. E.
Angus
,
W. D.
Kim
,
N. S.
Wang
,
M.
King
,
H.
Ghezzo
,
M. G.
Cosio
.
1985
.
Destructive index: a measurement of lung parenchymal destruction in smokers.
Am. Rev. Respir. Dis.
131
:
764
769
.
39
Finlay
,
G. A.
,
L. R.
O’Driscoll
,
K. J.
Russell
,
E. M.
D’Arcy
,
J. B.
Masterson
,
M. X.
FitzGerald
,
C. M.
O’Connor
.
1997
.
Matrix metalloproteinase expression and production by alveolar macrophages in emphysema.
Am. J. Respir. Crit. Care Med.
156
:
240
247
.
40
Zaynagetdinov
,
R.
,
T. P.
Sherrill
,
P. L.
Kendall
,
B. H.
Segal
,
K. P.
Weller
,
R. M.
Tighe
,
T. S.
Blackwell
.
2013
.
Identification of myeloid cell subsets in murine lungs using flow cytometry.
Am. J. Respir. Cell Mol. Biol.
49
:
180
189
.
41
Taraseviciene-Stewart
,
L.
,
N. F.
Voelkel
.
2008
.
Molecular pathogenesis of emphysema.
J. Clin. Invest.
118
:
394
402
.
42
Haq
,
I.
,
G. E.
Lowrey
,
N.
Kalsheker
,
S. R.
Johnson
.
2011
.
Matrix metalloproteinase-12 (MMP-12) SNP affects MMP activity, lung macrophage infiltration and protects against emphysema in COPD.
Thorax
66
:
970
976
.
43
Su
,
B. H.
,
Y. L.
Tseng
,
G. S.
Shieh
,
Y. C.
Chen
,
P.
Wu
,
A. L.
Shiau
,
C. L.
Wu
.
2016
.
Over-expression of prothymosin-α antagonizes TGFβ signalling to promote the development of emphysema.
J. Pathol.
238
:
412
422
.
44
Feinberg
,
M. W.
,
M. K.
Jain
,
F.
Werner
,
N. E.
Sibinga
,
P.
Wiesel
,
H.
Wang
,
J. N.
Topper
,
M. A.
Perrella
,
M. E.
Lee
.
2000
.
Transforming growth factor-beta 1 inhibits cytokine-mediated induction of human metalloelastase in macrophages.
J. Biol. Chem.
275
:
25766
25773
.
45
Massagué
,
J.
2012
.
TGFβ signalling in context.
Nat. Rev. Mol. Cell Biol.
13
:
616
630
.
46
Werner
,
F.
,
M. K.
Jain
,
M. W.
Feinberg
,
N. E.
Sibinga
,
A.
Pellacani
,
P.
Wiesel
,
M. T.
Chin
,
J. N.
Topper
,
M. A.
Perrella
,
M. E.
Lee
.
2000
.
Transforming growth factor-beta 1 inhibition of macrophage activation is mediated via Smad3.
J. Biol. Chem.
275
:
36653
36658
.
47
Barnes
,
P. J.
2007
.
Chronic obstructive pulmonary disease: a growing but neglected global epidemic.
PLoS Med.
4
:
e112
.
48
Orthgiess
,
J.
,
M.
Gericke
,
K.
Immig
,
A.
Schulz
,
J.
Hirrlinger
,
I.
Bechmann
,
J.
Eilers
.
2016
.
Neurons exhibit Lyz2 promoter activity in vivo: implications for using LysM-Cre mice in myeloid cell research.
Eur. J. Immunol.
46
:
1529
1532
.
49
Bies
,
J.
,
M.
Sramko
,
J.
Fares
,
M.
Rosu-Myles
,
S.
Zhang
,
R.
Koller
,
L.
Wolff
.
2010
.
Myeloid-specific inactivation of p15Ink4b results in monocytosis and predisposition to myeloid leukemia.
Blood
116
:
979
987
.
50
Chauhan
,
D.
,
P.
Pandey
,
T.
Hideshima
,
S.
Treon
,
N.
Raje
,
F. E.
Davies
,
Y.
Shima
,
Y. T.
Tai
,
S.
Rosen
,
S.
Avraham
, et al
.
2000
.
SHP2 mediates the protective effect of interleukin-6 against dexamethasone-induced apoptosis in multiple myeloma cells.
J. Biol. Chem.
275
:
27845
27850
.
51
Yang
,
W.
,
L. D.
Klaman
,
B.
Chen
,
T.
Araki
,
H.
Harada
,
S. M.
Thomas
,
E. L.
George
,
B. G.
Neel
.
2006
.
An Shp2/SFK/Ras/Erk signaling pathway controls trophoblast stem cell survival.
Dev. Cell
10
:
317
327
.
52
Cai
,
Z.
,
D. L.
Simons
,
X. Y.
Fu
,
G. S.
Feng
,
S. M.
Wu
,
X.
Zhang
.
2011
.
Loss of Shp2-mediated mitogen-activated protein kinase signaling in Muller glial cells results in retinal degeneration.
Mol. Cell. Biol.
31
:
2973
2983
.
53
Saxton
,
T. M.
,
T.
Pawson
.
1999
.
Morphogenetic movements at gastrulation require the SH2 tyrosine phosphatase Shp2.
Proc. Natl. Acad. Sci. USA
96
:
3790
3795
.
54
Qu
,
C. K.
,
W. M.
Yu
,
B.
Azzarelli
,
G. S.
Feng
.
1999
.
Genetic evidence that Shp-2 tyrosine phosphatase is a signal enhancer of the epidermal growth factor receptor in mammals.
Proc. Natl. Acad. Sci. USA
96
:
8528
8533
.
55
Lakso
,
M.
,
B.
Sauer
,
B.
Mosinger
Jr.
,
E. J.
Lee
,
R. W.
Manning
,
S. H.
Yu
,
K. L.
Mulder
,
H.
Westphal
.
1992
.
Targeted oncogene activation by site-specific recombination in transgenic mice.
Proc. Natl. Acad. Sci. USA
89
:
6232
6236
.
56
Kühn
,
R.
,
F.
Schwenk
,
M.
Aguet
,
K.
Rajewsky
.
1995
.
Inducible gene targeting in mice.
Science
269
:
1427
1429
.
57
Hogg
,
J. C.
,
R. M.
Senior
.
2002
.
Chronic obstructive pulmonary disease - part 2: pathology and biochemistry of emphysema.
Thorax
57
:
830
834
.
58
Wert
,
S. E.
,
M.
Yoshida
,
A. M.
LeVine
,
M.
Ikegami
,
T.
Jones
,
G. F.
Ross
,
J. H.
Fisher
,
T. R.
Korfhagen
,
J. A.
Whitsett
.
2000
.
Increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein D gene-inactivated mice.
Proc. Natl. Acad. Sci. USA
97
:
5972
5977
.
59
Zhang
,
X.
,
P.
Shan
,
G.
Jiang
,
L.
Cohn
,
P. J.
Lee
.
2006
.
Toll-like receptor 4 deficiency causes pulmonary emphysema.
J. Clin. Invest.
116
:
3050
3059
.
60
Lagente
,
V.
,
C.
Le Quement
,
E.
Boichot
.
2009
.
Macrophage metalloelastase (MMP-12) as a target for inflammatory respiratory diseases.
Expert Opin. Ther. Targets
13
:
287
295
.
61
Maeno
,
T.
,
A. M.
Houghton
,
P. A.
Quintero
,
S.
Grumelli
,
C. A.
Owen
,
S. D.
Shapiro
.
2007
.
CD8+ T cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice.
J. Immunol.
178
:
8090
8096
.
62
Chen
,
K.
,
D. A.
Pociask
,
J. P.
McAleer
,
Y. R.
Chan
,
J. F.
Alcorn
,
J. L.
Kreindler
,
M. R.
Keyser
,
S. D.
Shapiro
,
A. M.
Houghton
,
J. K.
Kolls
,
M.
Zheng
.
2011
.
IL-17RA is required for CCL2 expression, macrophage recruitment, and emphysema in response to cigarette smoke.
PLoS One
6
:
e20333
.

The authors have no financial conflicts of interest.

Supplementary data