Matrix metalloproteinase activity in the lung is increased in Hermansky-Pudlak syndrome

Wild-type, HPS1, and HPS2 mice (C57B/6 J, 8–10 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed in a pathogen-free animal facility at Thomas Jefferson University. HPS1 mice have homozygous mutation of the Hps1 gene, which encodes for a protein called BLOC-3, and HPS2 mice have homozygous mutation in the adaptor protein 3b1 (Ap3b1) gene, which is a subunit of the AP-3 protein complex. In general, HPS mice are phenotypically normal, except for a light coat appearance. HPS 1 and 2 mice also have large lamellar bodies in the alveolar epithelial type II cells of their lungs. Both strains of mice are also exquisitely sensitive to bleomycin. Throughout the study period, wild-type and HPS mice were maintained on a standard chow diet (13.5% calories from fat, 58% from carbohydrates, and 28.5% from protein) and permitted to feed ad libitum. Prior to the initiation of any study, the Institutional Animal Care and Use Committee at Thomas Jefferson University approved all animal protocols.

The diagnosis of HPS was established based on published criteria [22, 23]. Healthy controls were individuals without any known lung disease. Age, gender and smoking history for subjects are listed in Additional file 1: Table S1. All patients provided written informed consent to protocols 95-HG-0193 (clinicaltrials.​gov NCT00001456) and 04-HG-0211 (clinicaltrials.​gov NCT00084305). All study protocols were approved by the Institutional Review Boards at Thomas Jefferson University and the National Human Genome Research Institute prior to the initiation of any studies. BAL was performed and samples were processed as previously described [24].

Lung injury was induced by instilling 0.025 U of bleomycin into the posterior oropharynx of anesthetized mice. Because HPS1 and HPS2 mice are more sensitive to bleomycin, lower doses of bleomycin were required for these investigations [2, 25, 26].

The activity of MMP-2 and -9 was assessed by gelatin zymography as previously described [8, 27]. Protein concentration was determined by Pierce™ BCA assay kit (Thermo Scientific, Rockford, IL). Murine and human BALF and lung homogenates were separated by electrophoresis using 10% SDS-polyacrylamide gels containing 0.1% gelatin. Gels were then washed in 2.5% triton 100 renaturing buffer followed by overnight incubation in developing buffer. To visualize bands gels were stained with 0.5% Coomassie blue for 1 h and then destained with 40% methanol/10% acetic acid until clear bands were visualized. Densitometry was performed as previously described and the MMPs activity were normalized for total BALF and lung homogenates protein concentration.

Gene transcript levels were quantified by real-time PCR as previously described [28]. In brief, RNA was isolated using RNeasy Mini-Kit (QIAGEN, Valencia, CA). All reactions were performed with 1 μM of forward and reverse primers along with SYBR Green I GoTaq qPCR Master Mix (Promega, Madison, WI). Primer sets were amplified using protocols previously described [28‐30]. All values were normalized to a control gene such as 18S.

Murine lung epithelial 12 (MLE12) cells were obtained from ATCC (Manassas, VA) and cultured as previously described [28, 29]. Cells were plated in 6-well plates with or without bleomycin (50 μg/ml) or Akt inhibitor (1 μM). After 24 h, supernatant was collected and centrifuged to remove cellular debris and then stored at − 80 °C. Whole cell lysates were also collected to measure transcript or protein levels.

pLKO.1-based lentiviral Ap3b1 shRNA constructs (RHS4533; clone ID, TRCN0000118642) were used to silence the AP3 gene in MLE12 cells in order to create cells reminiscent to those in the lungs of HPS-2 patients. Scrambled shRNAs were used as a control. Lentiviral transductions for both Ap3b1 and scrambled control were performed as previously described [28].

Protein concentration was determined by Pierce™ BCA assay kit (Thermo Scientific, Rockford, IL). Aliquots of protein lysates were transferred onto nitrocellulose membranes and then blocked with the Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE) for 1 h at RT. This step was followed by an incubation step with a specific polyclonal rabbit primary antibody directed against MMP-2, MMP-9, Akt, phosphorylated Akt, or β-actin (Sigma-Aldrich, St. Louis, MO). Next, membranes were incubated in a solution containing a donkey anti-rabbit or anti-mouse antibody (Li-Cor Biosciences, Lincoln, NE). After three sequential washes with PBS, immunoblots were visualized using the Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE).

To assess whether HPS alters MMP levels in the lung, we first performed quantitative PCR to evaluate transcript levels for several different MMPs known to be expressed in the mouse lung and which have also been linked to lung disease, including Mmp-2, − 3, − 7, − 8, − 9, − 12 and − 14. As demonstrated in Fig. 1a, we found that transcript levels for each of the Mmps evaluated were readily detectable in the lungs of wild-type mice and that levels for most, if not all, Mmps were upregulated in the lungs of HPS2 mice. However, only transcript levels for Mmp-2 and -9 were found to be significantly increased (p-value < 0.05) relative to controls, and only levels of Mmp-2 and Mmp-9 were increased by more than 2-fold. Consistent with the marked upregulation in Mmp-2 and Mmp-9 expression, we found that protein levels and enzymatic activity for each of these enzymes were dramatically increased in whole lung tissue digests of HPS2 mice (Fig. 1b, c). In contrast, only levels and enzymatic activity of MMP-2 were increased in BALF (Fig. 1d, e). Altogether, these findings indicate that expression and activity of MMPs, especially the gelatinases MMP-2 and MMP-9, are increased in the lung of HPS mice.

Because MMPs are produced by many different cell types, we next sought to localize the expression of MMP-2 and -9 in the HPS2 mouse lung. As shown in Fig. 2a, immunostaining for MMP-2 and -9 did not detect significant protein expression in the lungs of wild-type mice. In contrast, we found that levels of both enzymes were readily detectable in the lungs of HPS2 mice and that staining was most abundant in AEC2 of the distal pulmonary epithelium, as judged by the cellular location and presence of lamellar bodies (vacuolated structures) in intensely stained cells (brown color) (Fig. 2a). Of note, lower intensity staining was also observed in other regions of HPS2 lung, including the pulmonary interstitium and alveolar macrophages, suggesting that mesenchymal cells might also contribute to elevated MMP levels in the lungs of these mice.

Because expression of MMP-2 and -9 was readily apparent in AEC2, we next sought to determine whether epithelial deficiency of the Ap3b1 gene could by itself increase the expression of MMP enzymes. To test this, we performed shRNA knockdown of the Ap3b1 gene in murine lung epithelial 12 (MLE12) cells, a cell line often used to model AEC2 in culture [28, 29]. Consistent with findings in vivo, we found that shRNA knockdown of Ap3b1 readily increased MMP-2 and -9 expression, as demonstrated by a greater than 2-fold increase in transcript levels and a nearly 50% increase in protein levels for both MMP enzymes (Fig. 2b, c).

Expression of MMPs is known to increase in response to pro-fibrotic pulmonary insults, leading us to examine whether levels of these enzymes were further dysregulated in the HPS2 lung after pulmonary challenge. To test this, we administered a one-time, low dose (0.025 U) of bleomycin into the oropharynx of wild-type and HPS2 mice. The decision to use a low-dose of bleomycin was based on the understanding that HPS mice are exquisitely sensitive to this genotoxic insult, and that higher doses are universally fatal [17, 18, 26]. Consistent with this being a mild pulmonary insult, we found that low-dose bleomycin had little to no effect on the expression of MMPs in the lungs of wild-type mice at day 7 after injury (data not shown). In contrast, transcript levels for all of the MMPs assessed were significantly increased in the lungs of HPS2 mice relative to injured wild-type controls (Fig. 3a). Moreover, elevated transcript levels were also associated a marked upregulation in protein expression (Fig. 3b, d) and a dramatic increase in enzymatic activity for MMP-2 and -9 in whole lung lysates and BAL fluid (Fig. 3c, e).

Since broadly inhibiting MMP activity has been associated with significant toxicity in numerous cancer studies [19], we sought to examine the effects of inhibiting an upstream regulator of MMP activity. This would also circumvent the need to simultaneously inhibit multiple MMP enzymes. Recent work has shown that MMP expression can be regulated by the enzyme Akt [21, 31, 32], leading us to hypothesize that dysregulation of Akt might contribute to altering MMP expression in the HPS lung. To test this hypothesis, we compared levels of total and phosphorylated forms of this enzyme in control and HPS tissues. Although we did not detect a significant increase in total Akt levels, the activated form of this enzyme was dramatically increased in whole lung digests of HPS2 mice at baseline and at 7 days after bleomycin. Similarly to whole lung tissues, we found that phosphorylated Akt levels were also increased in AP3 deficient lung epithelial cells (Fig. 4b) at baseline and at 24 h after bleomycin exposure (Fig. 4c) and that this associated with an upregulation in MMP-2 and -9 expression (Fig. 4d). In order to determine whether Akt regulates MMP expression, we exposed cells to a pharmacological inhibitor of Akt to examine the effects on MMP levels. As shown in Fig. 4e, we found that pharmacological inhibition of Akt significantly reduced MMP levels in bleomycin-exposed cells, supporting the notion that chronic activation of Akt contributes to elevated MMP expression in the HPS lung.

Next, to determine whether MMP expression is dysregulated in other HPS models, we measured transcript levels for various MMPs in the lungs of HPS1 mice. Strikingly, we detected a marked upregulation in transcript levels for multiple MMPs in the lungs of HPS1 mice, including Mmp-2 and -9 as well as the Mmps 3, − 8, − 12 and − 14 (Fig. 5a). Similarly to HPS2 mice, we also found that protein levels for MMP-2 and -9 were increased in whole lung digests and that MMP-2 gelatinase activity was increased in the BALF fluid relative to age-matched controls (Fig. 5b, c). Likewise, transcript levels for MMPs were also dramatically increased in the lungs of HPS1 mice after bleomycin (Fig. 5d), and this associated with elevated MMP-2 and -9 protein levels in whole lung tissue digests (data not shown) and BALF (Fig. 5e).

Finally, to determine whether findings in mouse models were relevant to human disease, we assessed whether levels or activity of MMPs were altered in the lungs of HPS patients. As shown in Fig. 6, we found that protein levels for both MMP-2 and -9 were significantly increased in BALF of HPS patients relative to controls. Moreover, this associated with a significant upregulation in MMP-2 activity (Fig. 7a), although MMP-9 activity did not significantly differ between control and HPS patients (Fig. 7b). Interestingly, neither levels of MMP-2 and -9 nor activity of MMP-2 associated with the presence or absence of fibrosis or measures of lung function, such as diffusing for carbon monoxide or forced vital capacity (Fig. 7c, d).