Effect of beta-agonists on LAM progression PNAS PLUS and treatment

Kang Lea,1,2, Wendy K. Steagalla,1,3, Mario Stylianoub, Gustavo Pacheco-Rodrigueza, Thomas N. Darlingc, Martha Vaughana,3, and Joel Mossa,3

aPulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; bOffice of Biostatistics Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and cDepartment of Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814

Contributed by Martha Vaughan, December 6, 2017 (sent for review April 6, 2017; reviewed by Kevin Brown and Geraldine Finlay) Lymphangioleiomyomatosis (LAM), a rare disease of women, is (11). Although LAM lesions were originally considered to rep- associated with cystic lung destruction resulting from the pro- resent a benign neoplasm, LAM is now accepted as a with liferation of abnormal smooth muscle-like LAM cells with muta- metastatic dissemination of cancer-like LAM cells (12). tions in the complex (TSC) genes TSC1 and/or LAM cells are characterized by mutations in the tuberous TSC2. The mutant genes and encoded proteins are responsible for sclerosis complex (TSC) TSC1 or TSC2 gene that encodes, re- activation of the mechanistic target of rapamycin (mTOR), which is spectively, hamartin and tuberin (13–15). TSC is a rare genetic inhibited by (rapamycin), a drug used to treat LAM. Patients disease that affects multiple organ systems and results from who have LAM may also be treated with bronchodilators for asthma- mutations in one of the same two TSC genes (16). Hamartin and like symptoms due to LAM. We observed stabilization of forced tuberin form a cytosolic complex with Tre2-Bub2-Cdc16 domain expiratory volume in 1 s over time in patients receiving sirolimus and family member 7 (TBC1D7) (17). This complex inhibits the long-acting beta-agonists with short-acting rescue inhalers compared mechanistic target of rapamycin (mTOR) pathway, a promoter with patients receiving only sirolimus. Because beta-agonists increase of through the GTPase-activating protein (GAP) cAMP and PKA activity, we investigated effects of PKA activation on activity of tuberin toward Ras homolog enriched in (Rheb) + − the mTOR pathway. Human skin TSC2 / fibroblasts or LAM lung cells (17). Rheb in its GTP-bound form is a critical activator of incubated short-term with isoproterenol (beta-agonist) showed a mTOR; tuberin converts active Rheb-GTP to inactive Rheb- CELL BIOLOGY sirolimus-independent increase in of S6, a down- GDP (18). stream effector of the mTOR pathway, and increased cell growth. Cells mTOR, a serine/ , is found in mTORC1 and incubated long-term with isoproterenol, which may lead to beta- mTORC2 complexes (19). mTORC1 comprises regulatory- adrenergic receptor desensitization, did not show increased S6 phos- associated protein of mTOR (Raptor), mammalian lethal with phorylation. Inhibition of PKA blocked the isoproterenol effect on Sec13 protein 8 (mLST8; also known as GβL), proline-rich AKT S6 phosphorylation. Thus, activation of PKA by beta-agonists increased substrate of 40 kDa (PRAS40), and DEP domain-containing phospho-S6 independent of mTOR, an effect abrogated by beta- mTOR-interacting protein (Deptor) (20). mTORC2 is also a agonist–driven receptor desensitization. In agreement, retrospective clinical data from patients with LAM suggested that a combination Significance of bronchodilators in conjunction with sirolimus may be preferable to sirolimus alone for stabilization of pulmonary function. Lymphangioleiomyomatosis (LAM) is a destructive lung disease driven by neoplastic LAM cells with a mutated tumor sup- cyclic AMP | sirolimus | lymphangioleiomyomatosis | pressor gene TSC1 or TSC2, leading to increased activity of the tuberous sclerosis complex | bronchodilators mechanistic target of rapamycin (mTOR), which is inhibited by sirolimus (rapamycin). Beta-agonists may treat asthma-like ymphangioleiomyomatosis (LAM), a rare multisystem dis- symptoms due to LAM. We observed stabilization of forced ex- Lease affecting primarily women, is characterized by cystic lung piratory volume in 1 s in patients receiving sirolimus and long- destruction, which can lead to respiratory failure, abdominal acting beta-agonists with short-acting rescue inhalers compared tumors [e.g., renal angiomyolipomas (AMLs)], and lymphatic with patients receiving only sirolimus. Human TSC2+/− skin fi- – involvement (e.g., lymphangioleiomyomas, adenopathy) (1 4). broblasts and LAM cells from explanted lungs treated with Depending on organ involvement, patients may exhibit pro- sirolimus and the short-term, but not long-term, beta-agonist gressive dyspnea on exertion, pneumothoraces, chylous pleural isoproterenol showed increased phospho-S6 levels and cell effusions, ascites, and abdominal hemorrhage (5). LAM is often growth due to activation of a cAMP/PKA-dependent pathway. mistakenly diagnosed as another respiratory disease, such as Long-acting beta-agonists affect phospho-S6 content, leading to asthma, emphysema, chronic bronchitis, or chronic obstructive stabilization of lung function in LAM patients. pulmonary disease (6–8). Many patients are treated with bron- chodilators to alleviate asthma-like symptoms due to LAM dis- Author contributions: K.L., W.K.S., G.P.-R., M.V., and J.M. designed research; K.L., W.K.S., ease. In fact, in a study of 235 patients who had LAM, about 49% and G.P.-R. performed research; T.N.D. contributed new reagents/analytic tools; K.L., W.K.S., and M.S. analyzed data; and K.L., W.K.S., G.P.-R., T.N.D., M.V., and J.M. wrote used bronchodilators regularly (9). the paper. LAM is characterized by proliferation of abnormal smooth Reviewers: K.B., National Jewish Health; and G.F., UpToDate/Wolters Kluwer. muscle-like LAM cells in the lung, resulting in parenchymal The authors declare no conflict of interest. cystic destruction. LAM cells are believed to proliferate in axial Published under the PNAS license. lymphatics and lung interstitium, leading to airway and lymphatic 1K.L. and W.K.S. contributed equally to this work. obstruction (2, 10). While LAM cells may be largely parenchy- 2Present address: Department of Biochemistry and Molecular Medicine, School of Medi- mal, Hayashi et al. (11) showed bronchial involvement by LAM cine and Health Sciences, The George Washington University, Washington, DC 20037. cells in explanted lungs of all 30 patients examined. A significant 3To whom correspondence may be addressed. Email: [email protected], vaughanm@ portion of these patients also had markers of chronic in- nih.gov, or [email protected]. flammation (e.g., mononuclear cell infiltration, goblet cell hy- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. perplasia, squamous cell metaplasia, thickening of basal lamina) 1073/pnas.1719960115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1719960115 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 multimer, sharing proteins, such as mLST8 and Deptor, with P = 0.0417; adjusted for initial DLCO, sirolimus treatment, and mTORC1, whereas the defining component of mTORC2 is time of visit). A statistical interaction was seen between use of Raptor-independent companion of mTOR (Rictor) (21, 22). The bronchodilators and sirolimus treatment, such that the effect of mTORC1 substrates [e.g., P70 S6 kinase (P70), 4E-binding bronchodilator use on pulmonary function was different in pa- protein 1 (4EBP1), unc-51–like -activating kinase 1 tients not receiving sirolimus compared with those being treated (ULK1)] regulate cell size, proliferation, and autophagy in a with the drug. In patients not receiving sirolimus, those tak- phosphorylation-dependent fashion (23). Activation of S6 ki- ing bronchodilators tended to have lower percent predicted nases by mTOR promotes phosphorylation of several substrates, FEV1 compared with those not using bronchodilators, whereas including ribosomal protein S6, eukaryotic initiation factor 4B subjects on sirolimus had the opposite pattern (P < 0.001). (eIF4B), programmed cell death 4 (PDCD4), eukaryotic elon- Subjects not on sirolimus and on bronchodilators had lower gation factor 2 kinase (eEF-2K), and S6K1 Aly/REF-like target percent predicted DLCO compared with those not using bron- (SKAR) (23). Phosphorylation of S6, a component of the 40S chodilators, whereas subjects on sirolimus had similar DLCO ribosomal subunit, is associated with increased protein synthesis regardless of bronchodilator use (P = 0.002). Overall, the rate of and cell proliferation (24). mTORC2 regulates metabolism and change of FEV1 was −1.187 ± 0.077 (mean ± SE) percent cytoskeletal organization by phosphorylating AGC , such predicted FEV1 per year in those without bronchodilator use as Akt and PKC (22, 25, 26). and −1.358 ± 0.104 with bronchodilator use, and the rate of The absence of functional tuberin leads to persistence of Rheb change of DLCO was −1.618 ± 0.065 percent predicted DLCO in its GTP-bound state with mTORC1 activation, as was observed per year without bronchodilator use and −1.563 ± 0.093 with in LAM lung lesions and AMLs. Sirolimus (rapamycin), bound bronchodilator use. These values are not significantly different. to FK506-binding protein 12 (FKBP12), interacts directly with Since the interaction of sirolimus treatment and bronchodila- mTORC1, inhibiting its kinase activity (27, 28), and is now fre- tor use was significant, we analyzed the use of bronchodilators by quently used to treat patients with moderate to severe pulmonary patients not receiving sirolimus separately from those receiving siro- LAM (29). In patients with LAM, sirolimus stabilized forced ex- limus. A total of 405 patients were not receiving sirolimus for piratory volume in 1 s (FEV1) (29); decreased levels of the serum 2,642 visits (Table S1). Patients averaged 6.5 ± 0.2 visits (range: 1–28) biomarker VEGF-D (30); and reduced the sizes of AMLs (31), with a follow-up time of 4.6 ± 0.2 y (range: 0.0–17.8). Patients chylous effusions, and lymphangioleiomyomas (32). In May 2015, reported no use of bronchodilators at 1,525 (57.7%) visits to the NIH sirolimus was approved by the US Food and Drug Administration and the use of bronchodilators at 1,117 (42.3%) visits. Beta-agonist for use in LAM, based on the results of the Multicenter Inter- use comprised 407 (36.4%) visits with reported bronchodilator use, national LAM Efficacy and Safety of Sirolimus Trial (29). followed by use of beta-agonists plus steroids (376 or 33.7% of visits), While sirolimus is the treatment of choice for patients with beta-agonists plus steroids plus anticholinergics (137 or 12.3% of LAM who have rapidly progressive disease, some patients respond visits), beta-agonists plus anticholinergics (121 or 10.8% of visits), and better than others (32). Since disease progression on sirolimus can be fewer than 30 visits of each of the other combinations of broncho- variable, we examined other pathways that might be involved in dilators. Use of bronchodilators was significantly associated with LAM disease progression. The cAMP/PKA pathway, as activated by FEV1 and DLCO (both P < 0.001; adjusted for initial FEV1 or chronic stress through beta2-adrenergic receptors, may be involved DLCO and time of visit). Patients with no bronchodilator use aver- in tumor progression and metastasis (33, 34). A significant fraction aged 70.1 ± 0.5 and 64.7 ± 0.4percentpredictedFEV1andDLCO, of patients with LAM have reversible airflow obstruction that is respectively, while those using bronchodilators averaged 66.9 ± 0.5 treated with short- or long-acting bronchodilators (9). A response to and 62.1 ± 0.4 percent predicted FEV1 and DLCO, respectively. bronchodilators was more common in patients with lung LAM Subjects not receiving sirolimus but using bronchodilators showed a nodules that line the lung cysts (2, 7, 9, 35), and was associated with faster decline in FEV1 than those not using bronchodilators (P < an accelerated decline in FEV1 (9, 35). Here, we found that brief 0.001; no bronchodilator use: −1.397 ± 0.070 vs. −1.9534 ± 0.111 + − incubation of human TSC2 / skin fibroblasts and LAM lung cells percent predicted FEV1 per year with bronchodilator use). The with the beta-agonist isoproterenol increased phosphorylation of yearly rate of decline of DLCO was also faster in patients using S6 via the cAMP/PKA pathway, independent of sirolimus. Short- bronchodilators (−2.009 ± 0.111 percent predicted DLCO per year) term incubation with a beta-agonist increased cell proliferation and versus those not using bronchodilators (−1.817 ± 0.070 percent phospho-S6 content more than prolonged incubation. These data predicted DLCO per year); however, this difference was not signifi- were consistent with our retrospective review of a longitudinal study cant (P = 0.143). of patients with LAM that showed the rate of FEV1 change may be The analysis of the effect of bronchodilators on pulmonary dependent on the type of beta-agonist used by the patient. function in patients receiving sirolimus included 108 patients with 460 visits (Table S2). Patients averaged 3.0 ± 0.2 y of Results sirolimus use (range: 0.08–10.45 y). Patients reported no use Retrospective Study of the Effects of Bronchodilators on Pulmonary of bronchodilators at 163 (35.4%) of visits to the NIH and use of Function. We have previously reported that a significant number bronchodilators at 297 (64.6%) visits. Use of beta-agonists plus of patients with LAM have partially reversible airflow obstruc- steroids comprised 99 (21.5%) of visits with reported broncho- tion, as measured by a positive bronchodilator response to dilator use, followed by beta-agonists (82 or 17.8% of visits) and nebulized albuterol, which was associated with a more rapid beta-agonists plus steroids plus anticholinergics (75 or 16.3% of decline in pulmonary function (9). We retrospectively analyzed visits). Use of bronchodilators in this group did not significantly the effect of bronchodilator use on pulmonary function in affect the rate of change of FEV1 or DLCO. 426 patients with sporadic LAM or LAM/TSC over 3,102 visits (Tables S1 and S2). Clinical records were examined for reported Retrospective Study of the Effects of Beta-Agonists on Pulmonary bronchodilator use by the patient. Bronchodilator groups in- Function. We next looked specifically at the effects of beta- cluded beta-agonists, steroids, anticholinergics, and other [e.g., agonists on pulmonary function by retrospectively analyzing leukotriene receptor antagonists, cyclic nucleotide phosphodi- beta-agonist use of 426 patients with sporadic LAM or LAM/ esterase inhibitors (PDEs), mast cell stabilizers] (Table S3). Use TSC over 3,080 visits (Tables S4 and S5). Use of beta-agonists of bronchodilators was not significantly associated with percent was significantly associated with percent predicted FEV1 (P = predicted FEV1 (P = 0.128; adjusted for initial FEV1, sirolimus 0.043; adjusted for initial FEV1, sirolimus treatment, time of visit, treatment, time of visit, and age) and was somewhat associated and age) and with DLCO (P = 0.006; adjusted for initial DLCO, with diffusing capacity of the lung for carbon monoxide (DLCO; sirolimus treatment, and time of visit). As with bronchodilator use, a

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1719960115 Le et al. Downloaded by guest on September 26, 2021 statistical interaction was seen between use of beta-agonists Table 2. Rate of change of FEV1 categorized by beta-agonist PNAS PLUS and sirolimus treatment, such that the effect of beta-agonist use subtype in patients receiving sirolimus on pulmonary function was different in patients not receiving FEV1 rate of change, mean sirolimus compared with those receiving sirolimus. Subjects not re- Beta-agonist type percent predicted per year ± SE ceiving sirolimus but being treated with beta-agonists tended to have lower FEV1 compared with those not using beta-agonists, whereas None −1.297 ± 0.371 (122 visits) subjects receiving sirolimus had the opposite pattern (P < 0.001). Beta-agonist 0.125 ± 0.288 (187 visits) Subjects not receiving sirolimus but using beta-agonists had lower P = 0.002 DLCO compared with those not using beta-agonists, whereas None −1.311 ± 0.346 (122 visits) subjects receiving sirolimus had similar DLCO regardless of beta- Short-acting −0.393 ± 0.665 (48 visits) agonist use (P = 0.004). Overall, the rate of change of FEV1 was Both short-acting and long-acting 0.493 ± 0.365 (104 visits) −1.198 ± 0.077 percent predicted FEV1 per year without beta- Long-acting 0.880 ± 0.782 (35 visits) agonist use and −1.322 ± 0.108 with beta-agonist use, and the N vs. S P = 0.2253 = rate of change of DLCO was −1.618 ± 0.065 percent predicted N vs. SL P 0.0004 = DLCO per year without beta-agonist use and −1.478 ± 0.098 with N vs. L P 0.0113 = beta-agonist use. These values are not significantly different. S vs. SL P 0.2399 = Since the interaction of sirolimus treatment and beta-agonist S vs. L P 0.2103 = use was significant, we analyzed the visits of patients not receiving SL vs. L P 0.6525 sirolimus separately from the visits of those on sirolimus. A total of With statistical correction, significant P values are those less than 0.0083. 404 patients were not on sirolimus for 2,620 visits (Table S4). Beta- L, long-acting beta-agonist; N, no beta agonist; S, short-acting beta-agonist; agonist groups included short-acting, long-acting, and both. Patients SL, short- and long-acting beta-agonists. reported no use of beta-agonists at 1,594 (60.8%) of visits to the NIH and the use of beta-agonists at 1,026 (39.2%) visits. Short- acting beta-agonist use comprised 408 (39.8%) of visits with short-acting beta-agonists (71 or 25.2% of visits) and long-acting reported beta-agonist use, followed by both (386 or 37.6% of visits) beta-agonists (57 or 20.2%). Use of beta-agonists in this group did not significantly affect the rate of change of FEV1 or DLCO (P = and long-acting beta-agonists (232 or 22.6% of visits). Use of beta- P = agonists was significantly associated with FEV1 and DLCO (both 0.415 and 0.053, respectively). CELL BIOLOGY P < 0.001; adjusted for initial FEV1 or DLCO and time of visit). Analysis of the Effect of Beta-Agonist Subtypes on FEV1 in Patients Patients with no beta-agonist use averaged 70.3 ± 0.4 and 65.0 ± 0.4 Not Receiving Sirolimus. A multivariate model predicting FEV1 and percent predicted FEV1 and DLCO, respectively, while those using ± ± adjusting for initial FEV1, time of visit, age, and type of beta-agonist beta-agonists averaged 66.9 0.5 and 62.2 0.4 FEV1 and DLCO, (none and long-acting, short-acting, or both) examining all of the respectively. Subjects not receiving sirolimus but using beta-agonists visits by patients not receiving sirolimus resulted in a significant (P < showed a faster decline in FEV1 than those not using beta-agonists P < − ± − ± 0.01) contribution of all variables considered. The rate of change of ( 0.001; no beta-agonist use: 1.470 0.070 vs. 1.939 0.118 FEV1 for those not using beta-agonists was −1.304 ± 0.082 percent percent predicted FEV1 per year with beta-agonist use). The yearly predicted per year, while that for short-acting beta-agonists rate of change of DLCO was not different in patients using beta- was −1.677 ± 0.238, that for both was −0.783 ± 0.255, and − ± agonists ( 1.791 0.125) versus those not using beta-agonists that for long-acting beta-agonists was −2.443 ± 0.391. The rate of − ± P = ( 1.732 0.081) ( 0.689). change of FEV1 of patients on long-acting beta-agonists was sig- The analysis of the effect of beta-agonists on pulmonary func- nificantly different from that of patients on both long-acting and tion in patients receiving sirolimus included 107 patients with short-acting beta-agonists (P < 0.001) and from that of patients not 460 visits (Table S5). Patients reported no use of beta-agonists at on beta-agonists (P = 0.004) (Table 1 and Table S6). 178 (38.7%) visits to the NIH and use of beta-agonists at 282 (61.3%) visits. Short- and long-acting beta-agonist use comprised Analysis of the Effect of Beta-Agonist Subtypes on FEV1 in Patients 154 (54.6%) of visits with reported beta-agonist use, followed by Receiving Sirolimus. Since 51 of the 107 patients on sirolimus only had one or two visits receiving sirolimus, we refined the dataset to exclude visits before 2010 and then those patients without at Table 1. Rate of change of FEV1 categorized by beta-agonist least three visits approximately 6 mo apart. A total of 53 patients subtype in patients not receiving sirolimus with 349 visits were included in this study; 22 of them had in- FEV1 rate of change, mean formation from visits while not receiving sirolimus, for a total of Beta-agonist type percent predicted per year ± SE 40 visits (Tables S7 and S8). The use of beta-agonists was not significantly associated with percent predicted FEV1 (P = 0.174) None −1.470 ± 0.070 (1,594 visits) or DLCO (P = 0.186) after adjusting for initial FEV1 or DLCO Beta-agonist −1.939 ± 0.118 (962 visits) and sirolimus treatment. In this study population, the interaction < P 0.001 of sirolimus treatment and beta-agonist use was not significant, − ± None 1.304 0.082 (1,594 visits) indicating that the effect of beta-agonists on FEV1 or DLCO was − ± Short-acting 1.677 0.238 (391 visits) not different in patients receiving or not receiving sirolimus − ± Both short-acting and long-acting 0.783 0.255 (349 visits) treatment. The use of beta-agonists had no significant effect on − ± Long-acting 2.443 0.391 (222 visits) the rate of change of DLCO (−0.761 ± 0.285 percent predicted = N vs. S P 0.1291 per year for no beta-agonist use versus −0.458 ± 0.222 percent = N vs. SL P 0.0457 predicted per year for those using beta-agonists; P = 0.391). = N vs. L P 0.0040 Beta-agonist use did significantly affect the yearly rate of change = S vs. SL P 0.0095 of FEV1, however, with a rate of −1.297 ± 0.371 percent pre- = S vs. L P 0.0908 dicted FEV1 per year for those not using beta-agonists (134 vis- = SL vs. L P 0.0003 its) versus 0.125 ± 0.288 for those using beta-agonists (215 visits) P = With statistical correction, significant P values are those less than 0.0083. ( 0.002). Use of beta-agonists may have stabilized the L, long-acting beta-agonist; N, no beta agonist; S, short-acting beta-agonist; FEV1 compared with that in patients not using beta-agonists, SL, short- and long-acting beta-agonists. who continue to decline.

Le et al. PNAS Early Edition | 3of10 Downloaded by guest on September 26, 2021 + − Fig. 1. Effects of isoproterenol (ISO) and 8-Br-cAMP on phosphorylation of S6 and P70 in human skin TSC2 / fibroblasts incubated without and with + − sirolimus (Siro). (A and D)HumanskinTSC2 / cells were incubated for 1 h with control (Con) or the indicated concentration of ISO or 8-Br-cAMP (cAMP) before analysis by Western blotting with indicated bodies and densitometric quantification. *P < 0.01vs.Con.(B and E) Cells were incubated for 1 h with DMSO or 20 or 200 nM Siro, followed by 1 h with additional 1 μM ISO or 1 mM cAMP before analysis of indicated proteins by Western blotting and densitometric quantification. (B)*P < 0.01 vs. Con; **P < 0.05 vs. DMSO; ***P < 0.01 vs. Siro. (E)*P < 0.05 vs. DMSO; **P < 0.05 vs. 20 nM Siro; ***P < 0.05 vs. 200 nM Siro. (C and F) After a 72-h incubation with nontargeted (NT) or TSC2 siRNA, TSC2+/− cells were incubated for an additional hour with DMSO or 200 nM Siro,followedby1hwithadditional1μM ISO or 1 mM cAMP before analysis of indicated proteins by Western blotting and densitometric quantification. (C)*P < 0.01 vs. Con + NT siRNA; #P < 0.005 vs. Con + TSC2 siRNA; $P < 0.01 vs. DMSO + TSC2 siRNA; **P < 0.05 vs. Siro + TSC2 siRNA. (F)*P < 0.05 vs. DMSO + NT siRNA; **P < 0.01 vs. Siro + NT siRNA; #P < 0.01 vs. DMSO + TSC2 siRNA; ***P < 0.05 vs. Siro + TSC2 siRNA.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1719960115 Le et al. Downloaded by guest on September 26, 2021 + − PNAS PLUS The type of beta-agonist was significantly associated with neous than LAM lung cultures. Incubation of human skin TSC2 / percent predicted FEV1 (P < 0.001) after adjusting for initial FEV1, fibroblasts with isoproterenol for 1 h increased phosphorylation of sirolimus treatment, and time of visit. Here again, the interaction of S6 and P70 in a concentration-dependent manner (Fig. 1A), with sirolimus treatment and type of beta-agonist used was significant (P < no effect on phospho-4EBP1 or phospho-ULK1 levels. Sirolimus 0.001), indicating that the effect of the different types of beta-agonists completely prevented the effects of isoproterenol on phospho-P70, was different in patients receiving or not receiving sirolimus. The type but only partially inhibited S6 phosphorylation (Fig. 1B), suggest- of beta-agonist was not a predictor of percent predicted FEV1 for ing that isoproterenol-stimulated phosphorylation of S6 was due to patients not receiving sirolimus after adjusting for initial FEV1 and a pathway in addition to mTORC1. As expected, since the TSC2 time of visit. In this study group, this analysis contained only 22 pa- gene product tuberin acted as a negative regulator of the tients with 40 visits (Table S7). The type of beta-agonist was a pre- mTORC1 pathway, levels of phospho-S6 and phospho-P70 were dictor of percent predicted FEV1 (P = 0.003) in patients receiving higher in cells transfected with TSC2 siRNA than with non- sirolimus treatment, after adjusting for initial FEV1 and time of visit. specific siRNA (36) (Fig. 1C). Interestingly, S6 phosphorylation This subpopulation included 53 patients with 309 visits receiving was still increased in cells depleted of TSC2 in the presence of sirolimus (Table S8): patients did not use beta-agonists for 122 sirolimus after incubation with isoproterenol (Fig. 1C). Beta- (39.5%) of these visits and used beta-agonists for 187 (60.5%) of agonists activate adenylyl cyclase, increasing formation of the these visits. The patients used short-acting beta-agonists for 25.7% of second messenger cAMP (37). Incubation of cells with the cAMP the beta-agonist visits, long-acting beta-agonists for 18.7%, and both analog 8-Br-cAMP also increased phosphorylation of S6 in the for 55.6%. Interestingly, for the patients receiving sirolimus treat- presence of sirolimus or in cells depleted of TSC2 (Fig. 1 D–F), ment, those with no beta-agonist use showed the fastest rate of de- consistent with the role of cAMP in the effects of isoproterenol. cline of FEV1 (−1.311 ± 0.346 percent predicted FEV1 per year), As seen with isoproterenol, the cAMP-dependent increase in compared with those using short-acting beta-agonists alone (−0.393 ± phospho-p70 was blocked by sirolimus. All data supported the 0.665 percent predicted FEV1 per year), those using long-acting beta- view that isoproterenol and cAMP altered S6 phosphorylation agonists alone (0.880 ± 0.782 percent predicted FEV1 per year), and via both mTOR-dependent and -independent pathways. those using both (0.493 ± 0.365 percent predicted FEV1 per year). We also explored effects of bronchodilators ipratropium, an anti- When comparing the rates of change of FEV1 pairwise, only the cholinergic drug, and Montelukast, a leukotriene receptor antagonist. comparison of no beta-agonist use versus use of both was significant Incubation with ipratropium for1hincreasedS6andP70phos- (P = 0.0004; the corrected level of significance is 0.0083) (Table 2). phorylation in a concentration-dependent manner. The effects on

These data suggest that the use of both short- and long-acting beta- phospho-S6, however, were completely blocked by sirolimus, sug- CELL BIOLOGY agonists stabilizes the rate of decline of FEV1 for patients undergoing gesting that effects on S6 phosphorylation were via an mTOR- sirolimus treatment. dependent pathway (Fig. S1 A and B). Montelukast caused no obvious change in phospho-S6 or phospho-P70 content (Fig. S1C). Phosphorylation of S6 and P70 in TSC2+/− Cells Was Increased by Isoproterenol or cAMP. Since pulmonary function was affected Effects of Isoproterenol or cAMP on S6 or P70 Phosphorylation in TSC2+/− by bronchodilators, and especially by beta-agonists, we decided Cells in the Presence of H89 or Following PKA Catalytic Subunit α to examine the effect of beta-agonists (represented by iso- Depletion. We hypothesized that effects of isoproterenol and proterenol) on the mTOR pathway. To establish experimental cAMP were mediated through PKA, which comprises two regula- + − conditions, we first studied skin TSC2 / cells (germline muta- tory and two catalytic subunits (38). To explore the role of PKA in tion TSC2 c.4830G > A, p.W1610*), which are more homoge- regulation of S6 phosphorylation, we used H89, a PKA inhibitor,

+ − + − Fig. 2. Effects of H89 or PKA Cα knockdown on phosphorylation of S6 and P70 in TSC2 / cells. (A) TSC2 / cells were incubated for 1 h with DMSO or 10 μM H89, followed by 1 h with addition of 1 μM isoproterenol (ISO) before analysis of indicated proteins by Western blotting and densitometric quantification. *P < 0.01 vs. DMSO; **P < 0.01 vs. DMSO + ISO. (B) After a 1-h incubation with DMSO, 10 μM H89, or 200 nM sirolimus (Siro), cells were incubated for 1 h with addition of 1 μM ISO before analysis of indicated proteins by Western blotting and densitometric quantification. *P < 0.05 vs. DMSO; **P < 0.005 vs. DMSO + ISO; ***P < 0.01 vs. Siro; #P < 0.01 vs. Siro + ISO. (C) After a 72-h incubation with vehicle alone (Mock) or with nontargeted (NT) or PKA Cα siRNA (PKA), cells were incubated for 1 h with 1 μM ISO before analysis by Western blotting with indicated antibodies and densitometric quantification. *P < 0.05 vs. NT siRNA; **P < 0.01 vs. NT siRNA + ISO. (D) After a 72-h depletion of PKA Cα, cells were incubated for 1 h with DMSO or 200 nM Siro, followed by a 1-h incubation with additional 1 μM ISO before analysis by Western blotting with indicated antibodies and densitometric quantification. *P < 0.01 vs. DMSO; **P < 0.05 vs. DMSO + ISO; ***P < 0.01 vs. Siro; #P < 0.05 vs. Siro + ISO. Fig. S3 shows the effects on 4EBP1 and ULK1.

Le et al. PNAS Early Edition | 5of10 Downloaded by guest on September 26, 2021 and PKA catalytic subunit α (Cα) siRNA. Incubation of cells with serum-starved for 72 h and then incubated with media that contained H89 blocked the effects of isoproterenol or cAMP on phospho- isoproterenol or cAMP that had been incubated with similar cells for S6 and phospho-p70, with or without sirolimus (Fig. 2 A and B and 24 h. The results illustrated in Fig. S6A indicate that isoproterenol or Fig. S2A). H89 also blocked basal phosphorylation of S6, 4EBP1, cAMP was still active after exposure to cells in medium for 24 h at and ULK1, although we questioned whether this was due to in- 37 °C. Cells lost responsiveness, however, to isoproterenol or cAMP hibition of PKA (Fig. 2 A and B and Figs. S2A and S3), since after exposure to the agents for 72 h at 37 °C (Fig. S6B). H89 inhibits several kinases (39). To assess more specifically the role of PKA, we knocked down a PKA catalytic subunit. In contrast Isoproterenol or cAMP Increased Phospho-S6 in Cells Cultured from to H89, incubation with PKA Cα siRNA blocked only the phos- Lungs of Patients with LAM. LAM lung cell cultures are mixtures of phorylation of S6 and p70 induced by isoproterenol or cAMP, and cells, which include both TSC2 WT and TSC2-negative cells. had no effect on phosphorylation of 4EBP1 and ULK1 (Fig. 2 C Incubation of LAM cell cultures with isoproterenol or cAMP for and D and Figs. S2B and S3). All data are consistent with a model in 1 h with or without sirolimus increased phospho-S6 content (Fig. which isoproterenol and cAMP regulate S6 phosphorylation via 4 A and C and Fig. S7). Incubation with isoproterenol, however, mTOR-dependent (mTORC1/P70/S6 pathway that is inhibited by did not increase phospho-P70 content (Fig. 4A). In addition, sirolimus) and -independent (cAMP/PKA pathway) pathways. incubation with isoproterenol or cAMP continuously for 3 d did not increase the resulting phospho-S6 content, as was observed + − Long-Term Incubation with Isoproterenol Did Not Increase Phospho- with the TSC2 / cells (Fig. 4 B and D). Furthermore, H89 S6 or Phospho-P70 in Human TSC2+/− Cells or TSC2-Depleted Cells. To blocked the effects of isoproterenol on S6 phosphorylation, + − mimic the effects of long-acting beta-agonists, human TSC2 / suggesting that cAMP-PKA, but not the mTOR pathway, was cells were incubated with isoproterenol or cAMP for up to 72 h. involved in the regulation of phospho-S6 by isoproterenol in Both agents failed to increase S6 or P70 phosphorylation after a cultured lung LAM cells, a result that differed from the effects + − 48-h incubation despite producing an early response (Fig. 3A and seen with skin TSC2 / cells (Fig. S8). Figs. S4 and S5). Results were similar in TSC2-depleted cells (Fig. 3B and Fig. S4). As shown in Fig. 3 C and D, after continuous Isoproterenol or cAMP Increased Phospho-S6 in HMB45-Positive Human incubation of cells with isoproterenol for 3 d, no effect on S6 or Lung LAM Cells. Incubation with isoproterenol or cAMP for 1 h in- p70 phosphorylation was observed with or without sirolimus, even creased the number of human lung LAM cells with high cytosolic in TSC2-depleted cells. However, when cells were treated with reactivity to anti–phospho-S6 antibodies (Fig. 5A and Fig. S9A). isoproterenol for 1 h each day for 3 d (mimicking short-acting Sirolimus almost completely abolished detection of phospho-S6. beta-agonists), S6 or p70 phosphorylation was increased, even in Sirolimus effects were reversed by a 1-h incubation with iso- the presence of sirolimus (Fig. 3 E and F), suggesting the presence proterenol or cAMP (Fig. 5A and Fig. S9A). To determine of a sirolimus-resistant or partially mTOR-dependent pathway. whether LAM cells among the cultured LAM lung cell mixture To exclude the possibility that isoproterenol or cAMP loses its were affected by isoproterenol or cAMP, we used antibodies that + − activity after 24 h in medium at 37 °C, human TSC2 / cells were discriminate between LAM and non-LAM cells. The monoclonal

+ − Fig. 3. Effects of long-term incubation of TSC2 / cells with isoproterenol (ISO) on phosphorylation of + − S6 and P70. (A) TSC2 / cells were incubated with 1 μM ISO for the indicated time before analysis by Western blotting with indicated antibodies and densitometric quantification. *P < 0.01 vs. control (Con). (B) After a 72-h knockdown of TSC2, cells were incubated with 1 μM ISO for the indicated time be- fore analysis of indicated proteins by Western blot- ting and densitometric quantification. *P < 0.005 vs. TSC2 siRNA. Cells were incubated with DMSO or sirolimus (Siro) for 3 d, with or without ISO (C)or with ISO for 1 h each day for 3 d (E) before analysis by Western blotting with indicated antibodies and densitometric quantification. *P < 0.01 vs. Con; **P < 0.005 vs. DMSO; ***P < 0.01 vs. Siro. After a 72-h knockdown of TSC2, cells were incubated with DMSO or 200 nM Siro for 3 d, with or without 1 μM ISO (D) or with 1 μM ISO for 1 h each day for 3 d (F) before analysis of indicated proteins by Western blotting and densitometric quantification. *P < 0.01 vs. Con + nontargeted (NT) siRNA; **P < 0.01 vs. Con + TSC2 siRNA; #P < 0.01 vs. DMSO + TSC2 siRNA; ***P < 0.01 vs. Siro + TSC2 siRNA. Fig. S4 shows the effects on 4EBP1 and ULK1.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1719960115 Le et al. Downloaded by guest on September 26, 2021 + − Isoproterenol Affected Proliferation of TSC2 / Cells. S6, which is PNAS PLUS phosphorylated by P70, is a component of the 40S ribosomal subunit and thought to be involved in the regulation of cell size and proliferation (41). Isoproterenol was reported to inhibit cancer cell proliferation and tumor growth, which are mediated by inhibition of ERK1/2 phosphorylation through the cAMP/ PKA pathway (42). In Fig. 6A, after transfection with nontarget + − or TSC2 siRNA, TSC2 / cells were incubated continuously for 3 d as indicated with DMSO, sirolimus, and/or isoproterenol. Consistent with the earlier report (42), isoproterenol inhibited + − + − proliferation of TSC2 / cells or TSC2 / cells depleted of TSC2 with siRNA after 3 d of incubation (Fig. 6A). In cells incubated CELL BIOLOGY

Fig. 4. Effects of isoproterenol (ISO) or 8-Br-cAMP on phosphorylation of S6 in cells cultured from explanted lungs of patients with LAM who un- derwent transplantation. (A and C) Cells grown from LAM lung explants were incubated for 1 h with DMSO or 200 nM sirolimus (Siro), followed by 1 h with 1 μM ISO or 1 mM cAMP before analysis of indicated proteins by Western blotting and densitometric quantification. *P < 0.05 vs. control (Con); **P < 0.01 vs. DMSO; ***P < 0.01 vs. Siro. (B and D) Cells were in- cubated with DMSO or 200 nM Siro for 3 d, with or without 1 μM ISO or 1 mM cAMP, before analysis by Western blotting with indicated antibodies. Fig. S7 shows the effects on 4EBP1 and ULK1.

antibody HMB45 reacted with LAM cells, recognizing Pmel17, a 100-kDa glycoprotein originally identified in human melanoma cells (40). In the LAM lung cell mixture, HMB45 reacted with cytoplasmic granules resembling immature melanosomes (40). Few positive cells were found, indicating a low percentage of LAM B Fig. 5. Effects of isoproterenol (ISO) on phosphorylation of S6 in HMB45- cells in the mixture (Fig. 5 ). Incubation with isoproterenol or positive lung LAM cells. (A and B) Lung LAM cells after a 1-h incubation with cAMP for 1 h increased phospho-S6 (green fluorescence) in DMSO or 200 nM sirolimus (Siro), followed by a 1-h incubation with 1 μM sirolimus-treated, HMB45-reactive cells (Fig. 5B and Fig. S9B). LAM ISO, were fixed and stained with indicated antibodies. After incubation with lung-cultured cells were then treated with isoproterenol or cAMP for DMSO or 200 nM Siro for 3 d, with or without 1 μM ISO (C) or with ISO for 1 h 3 d either continuously or for 1 h each day. Continued incubation of each day for 3 d (D), cells were fixed, reacted with rabbit anti–phospho- the mixed culture with isoproterenol or cAMP for 3 d did not result in S6 polyclonal antibodies and mouse HMB45 monoclonal antibodies, and then prepared for confocal immunofluorescence microscopy. (Scale bar: increased phospho-S6 content, whereas incubation with isoproterenol 10 μm.) Mean fluorescence intensity of phospho-S6 in outlined area of each for 1 h each day increased phospho-S6 in HMB45-reactive cells (Fig. 5 cell (>50 in each population) was measured in pixels by ImageJ software C and D and Fig. S9 C and D), consistent with effects observed in (NIH). *P < 0.01 vs. DMSO; **P < 0.05 vs. Siro. Similar results were obtained +/− human skin TSC2 cells from patients with TSC. from three independent experiments.

Le et al. PNAS Early Edition | 7of10 Downloaded by guest on September 26, 2021 Fig. 6. Effects of isoproterenol (ISO), sirolimus (Siro), and TSC2 depletion on cell proliferation. (A) After transfection with nontargeted (NT) or TSC2 siRNA for 72 h, followed by incubation with DMSO, 200 nM Siro, and/or 1 μM ISO for the indicated days, human TSC2+/− cells were trypsinized and counted by microscopy. + − (B) After transfection with NT or TSC2 siRNA for 72 h, TSC2 / cells were incubated with DMSO or 200 nM Siro for the indicated days, along with 1 μM ISO for 1 h each day. Cells were counted as in A.*P < 0.05 vs. DMSO; #P < 0.05 vs. Siro. Similar results were obtained from three independent experiments.

+ − with isoproterenol for 1 h each day for 3 d, the inhibitory effects mus on human skin TSC2 / cell proliferation and S6 phosphoryla- of isoproterenol on cell proliferation were much less than those tion could be bypassed by short-term incubation of cells with after 3 d of continuous isoproterenol exposure (Fig. 6B). In- isoproterenol (Figs. 1 and 6). We also observed that the short-term hibition of cell proliferation by sirolimus was reduced when effects of isoproterenol or cAMP on HMB45-reactive cells among coupled with isoproterenol (Fig. 6B). We postulate that these those grown from lungs of LAM patients bypassed mTORC1 in- effects may have resulted from the increase in phospho-S6 by hibition by sirolimus (Fig. 5). These data showed that the cAMP/ short-term exposure to isoproterenol, which led to increased PKA pathway may play a critical role in LAM cell growth and cell proliferation. suggest that bronchodilators working through cAMP may affect LAM cell phospho-S6 content and proliferation, and thereby Discussion disease progression (Fig. 7). + − The past two decades have seen significant advances in LAM Human TSC2 / cells incubated with isoproterenol for 1 h had research and patient care, including both diagnosis and treat- markedly increased phosphorylation of P70 S6K and S6 (Fig. 1), ment (4). In 2000, TSC2 gene mutations in LAM cells were consistent with prior reports that cAMP/PKA can activate the reported (14), and increased mTOR activity was attributed to the mTOR pathway (43–45). cAMP/PKA directly phosphorylates dysregulated growth of these smooth muscle-like cells (36). mTOR and RAPTOR, which leads to the activation of P70 S6K Thus, activation of mTORC1 was postulated to be crucial for (46). In our study, only the increase of phospho-P70, not phospho- LAM cell growth. Consistent with these data, lung function of S6, was completely blocked by sirolimus, which indicated that patients was stabilized by sirolimus treatment (29). In some isoproterenol increased phospho-S6 via other pathways, in addi- cases, however, lung function continued to decline even in the tion to that through mTOR-P70 S6K. presence of sirolimus (32), suggesting that other pathways, in addition We then used H89 to show that the cAMP/PKA pathway was to mTOR, can regulate LAM cell growth. Many patients who have involved in phospho-S6 formation. However, phospho-4EBP1 LAM use bronchodilators, such as beta-adrenergic agents affecting and phospho-ULK1 levels were also reduced by H89, which the cAMP/PKA pathway, to treat airway obstruction. We questioned might reflect a nonspecific inhibitory effect. H89 blocks PKA whether disease progression both with and without sirolimus treat- actions through competitive inhibition of the ATP site on the ment was affected by use of bronchodilators. We found that patients PKA catalytic subunit (47–49). H89 inhibited at least eight other using the combination of short- and long-acting beta-agonists without kinases (e.g., MAPKAP-K1b, MSK1, KBα, SGK, S6K1, ROCK sirolimus had the slowest rate of decline in FEV1 (Table 1), while II, AMPK, CHK1) (39), thus potentially exhibiting a rela- those taking both sirolimus and short- and long-acting beta-agonists tively large number of PKA-independent effects. The fact that enjoyed a stabilization of FEV1 compared with patients on sirolimus H89 inhibited S6K1 and PKA (48) is consistent with the data in alone (Table 2). To determine why patients respond to these agents, Fig. 2A, in which H89 decreased the basal phosphorylation of S6. + − we tested isoproterenol, a nonselective beta-agonist, on TSC2 / and Because H89 is a somewhat nonspecific kinase inhibitor, we TSC2-deficient cells. We showed that the inhibitory effects of siroli- used siRNA-induced knockdown of a PKA catalytic subunit to

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1719960115 Le et al. Downloaded by guest on September 26, 2021 cAMP hydrolysis (60–62). With increased time of exposure to PNAS PLUS cAMP, PKA-mediated PDE phosphorylation and activation result in negative feedback regulation of cAMP signaling, by decreasing cAMP levels (63, 64). This offers another possible explanation of why prolonged incubation with isoproterenol led to loss of its effect on S6 or P70 phosphorylation. Our data suggest that short-term, but not long-term, in- cubation with beta-agonists increased phospho-S6 levels and cell growth. These results may provide an explanation of why patients taking both short- and long-acting beta-agonists had stabilization of FEV1 compared with patients not taking beta-agonists. In- haled bronchodilators have multiple potential targets in the lungs, including LAM cells, airway smooth muscle cells, mast cells, endothelial cells, eosinophils, neutrophils, macrophages, T lymphocytes, and type I and II pneumocytes (65). Each cell type may have a different response to a bronchodilator (e.g., differences in receptor desensitization, effects on mTOR as shown here in LAM lung cells versus skin cells) (65). In addition, many patients may take morethanonetypeofbronchodilator. Patients with asthma on long- acting beta-agonists receive steroids as well to control inflammation (66). Interestingly, patients not receiving sirolimus but taking long- acting beta-agonists showed a faster rate of decline in FEV1 than patients using both short- and long-acting beta-agonists. A total of 73.9% of the visits with both short- and long-acting beta-agonist use also included steroid use, while only 55.4% of the long-acting beta- Fig. 7. Proposed model for regulation of S6 phosphorylation (P) by the agonist visits also included steroid use. Thus, this result may be due to cAMP/PKA pathway. PKA, after activation by cAMP, regulates P of S6 via two the addition of steroids to the bronchodilator therapy. While this CELL BIOLOGY different routes. One, involving the mTORC1/P70/S6 pathway, is inhibited by pattern is even greater in the study of beta-agonist subtype in patients sirolimus, and the other, bypassing mTOR, involves P of S6 by PKA. R, reg- receiving sirolimus (87.5% of use of short- and long-acting beta- ulatory subunit. agonists is in conjunction with steroids versus 42.9% for long-acting beta-agonists plus steroids), the total number of long-acting beta- agonistvisits(35visits)isfarfewerthanthatofbothshort-and confirm a role of PKA. PKA is a heterotetramer comprising two long-acting beta-agonists (104 visits) and may not accurately reflect regulatory subunits and two catalytic subunits. Three catalytic sub- the effect on pulmonary function of long-acting beta-agonists alone. units have been identified, designated Cα,Cβ,andCγ (49–52). Cα Late-stage LAM lungs have been shown to have chronic inflamma- and Cβ are expressed in most tissues, whereas Cγ is expressed only in testis (52, 53). Cα is thought to be the predominant isoform, and its tion (11), and so a combination of beta-agonists to reduce airway deletion resulted in phenotypic changes in mice. Targeted deletion of obstruction plus a corticosteroid may be appropriate in some cases of PRKACA caused growth retardation and sperm dysfunction. In LAM. Chronic inflammation in chronic obstructive pulmonary dis- contrast, deletion of Cβ resulted in phenotypically normal mice (54). ease may be resistant to corticosteroids (67); peripheral blood PRKACA mutation was also found in cortisol-producing adrenocor- mononuclear cells from these patients showed increased mTOR tical adenomas (55). Our experiments with PKA Cα siRNA showed activity (68). Treatment of these cells with sirolimus restored sensi- that the cAMP/PKA pathway was involved in the regulation of tivity to corticosteroids (68), showing that the choice of bronchodi- S6 phosphorylation (Fig. 2), consistent with a previous report that lator to use with sirolimus may be important. PKA is an S6 kinase (56). We have shown here that beta-agonists affect phosphorylation In contrast to data from a 1-h short-term incubation, no effects of S6 and proliferation of LAM cells in both the presence and of a beta-agonist on phospho-S6, phospho-P70, phospho-4EBP1, absenceofsirolimus.Wehaveshown in a retrospective study that + − or phospho-ULK1 were seen when TSC2 / cells or LAM lung the type of beta-agonist used may affect the stability of pulmonary cells were incubated with isoproterenol continuously for 3 d. We function over time. Since use of a bronchodilator is important for hypothesize that this effect is due to desensitization of the re- the quality of life of a patient who has LAM, selection of a bron- ceptor and perhaps other components in the chodilator, such as a short-acting beta-agonist versus long-acting pathway (e.g., cyclic nucleotide PDEs as noted below). Among beta-agonist, or use of alternative bronchodilator therapy, including pathways leading to desensitization, PKA and G protein-coupled steroids, should be evaluated in a controlled . receptor kinase phosphorylate, and thus induce, the internal- ization of the beta-adrenergic receptor (57, 58). After more Materials and Methods prolonged agonist exposure, a net loss of cellular receptors Sources of antibodies and reagents are given in SI Materials and Methods, occurs through mechanisms independent of receptor phos- along with details of the study population, pulmonary function testing, and phorylation, such as ubiquitination, which results in receptor statistical analysis. The protocol was approved by the National Heart, Lung, degradation (59). This model (Fig. 7) could explain our data and Blood Institute Institutional Review Board (NHLBI protocols 95-H-0186 and showing that 1-h short-term incubation each day with iso- 96-H-0100), and written informed consent was obtained from all participants. +/− proterenol increased S6 phosphorylation, whereas the modifi- Culture of human TSC2 skin fibroblasts and LAM cells from explanted lungs, cation was not seen after a 3-d continuous, long-term incubation as well as confocal immunofluorescence microscopy and Western blotting, was (Figs. 3 and 5). performed as described in SI Materials and Methods. Increased phospho-S6 and -P70 was not observed following ACKNOWLEDGMENTS. We thank Drs. Daniela Malide and Christian Combs long-term incubation with 8-Br-cAMP, which bypassed the beta- (Light Microscopy Core Facility, National Heart, Lung, and Blood Institute) adrenergic receptor. An increase in cAMP produced activation of for their assistance with confocal microscopy. This research was supported by several PDEs, such as PDE3A, PDE3B, and PDE4s, which catalyze the Intramural Research Program, NIH, NHLBI.

Le et al. PNAS Early Edition | 9of10 Downloaded by guest on September 26, 2021 1. Matsui K, et al. (2000) Extrapulmonary lymphangioleiomyomatosis (LAM): Clinico- 36. Goncharova EA, et al. (2002) Tuberin regulates p70 S6 kinase activation and ribosomal pathologic features in 22 cases. Hum Pathol 31:1242–1248. protein S6 phosphorylation. A role for the TSC2 in pulmonary 2. Ferrans VJ, et al. (2000) Lymphangioleiomyomatosis (LAM): A review of clinical and lymphangioleiomyomatosis (LAM). J Biol Chem 277:30958–30967. morphological features. J Nippon Med Sch 67:311–329. 37. Wallukat G (2002) The beta-adrenergic receptors. Herz 27:683–690. 3. McCormack FX (2008) Lymphangioleiomyomatosis: A clinical update. Chest 133: 38. Francis SH, Corbin JD (1999) Cyclic nucleotide-dependent protein kinases: Intracellular 507–516. receptors for cAMP and cGMP action. Crit Rev Clin Lab Sci 36:275–328. 4. Taveira-DaSilva AM, Moss J (2014) Management of lymphangioleiomyomatosis. 39. Davies SP, Reddy H, Caivano M, Cohen P (2000) Specificity and mechanism of action of F1000Prime Rep 6:116. some commonly used protein kinase inhibitors. Biochem J 351:95–105. 5. Taveira-DaSilva AM, Steagall WK, Moss J (2006) Lymphangioleiomyomatosis. Cancer 40. Matsumoto Y, et al. (1999) Markers of cell proliferation and expression of mela- Contr 13:276–285. nosomal antigen in lymphangioleiomyomatosis. Am J Respir Cell Mol Biol 21: 6. Taylor JR, Ryu J, Colby TV, Raffin TA (1990) Lymphangioleiomyomatosis. Clinical 327–336. course in 32 patients. N Engl J Med 323:1254–1260. 41. Magnuson B, Ekim B, Fingar DC (2012) Regulation and function of ribosomal protein – 7. Kitaichi M, Nishimura K, Itoh H, Izumi T (1995) Pulmonary lymphangioleiomyoma- S6 kinase (S6K) within mTOR signalling networks. Biochem J 441:1 21. tosis: A report of 46 patients including a clinicopathologic study of prognostic factors. 42. Pérez Piñero C, Bruzzone A, Sarappa MG, Castillo LF, Lüthy IA (2012) Involvement of α β Am J Respir Crit Care Med 151:527–533. 2- and 2-adrenoceptors on breast cancer cell proliferation and tumour growth – 8. Chu SC, et al. (1999) Comprehensive evaluation of 35 patients with lymphangioleio- regulation. Br J Pharmacol 166:721 736. myomatosis. Chest 115:1041–1052. 43. Kwon G, Marshall CA, Pappan KL, Remedi MS, McDaniel ML (2004) Signaling elements 9. Taveira-DaSilva AM, et al. (2009) Reversible airflow obstruction in lymphangioleio- involved in the metabolic regulation of mTOR by nutrients, incretins, and growth factors in islets. 53:S225–S232. myomatosis. Chest 136:1596–1603. 44. Blancquaert S, et al. (2010) cAMP-dependent activation of mammalian target of ra- 10. Johnson SR, et al.; Review Panel of the ERS LAM Task Force (2010) European Re- pamycin (mTOR) in thyroid cells. Implication in mitogenesis and activation of CDK4. spiratory Society guidelines for the diagnosis and management of lymphangioleio- Mol Endocrinol 24:1453–1468. myomatosis. Eur Respir J 35:14–26. 45. de Joussineau C, et al. (2014) mTOR pathway is activated by PKA in adrenocortical 11. Hayashi T, et al. (2016) Bronchial involvement in advanced stage lymphangioleio- cells and participates in vivo to apoptosis resistance in primary pigmented nodular myomatosis: Histopathologic and molecular analyses. Hum Pathol 50:34–42. adrenocortical disease (PPNAD). Hum Mol Genet 23:5418–5428. 12. McCormack FX, Travis WD, Colby TV, Henske EP, Moss J (2012) Lymphangioleio- 46. Liu D, et al. (2016) Activation of mTORC1 is essential for β-adrenergic stimulation of myomatosis: Calling it what it is: A low-grade, destructive, metastasizing neoplasm. adipose browning. J Clin Invest 126:1704–1716. Am J Respir Crit Care Med 186:1210–1212. 47. Engh RA, Girod A, Kinzel V, Huber R, Bossemeyer D (1996) Crystal structures of cat- 13. Smolarek TA, et al. (1998) Evidence that lymphangiomyomatosis is caused by alytic subunit of cAMP-dependent protein kinase in complex with iso- TSC2 mutations: Chromosome 16p13 loss of heterozygosity in angiomyolipomas and quinolinesulfonyl protein kinase inhibitors H7, H8, and H89. Structural implications – lymph nodes from women with lymphangiomyomatosis. Am J Hum Genet 62:810 815. for selectivity. J Biol Chem 271:26157–26164. 14. Carsillo T, Astrinidis A, Henske EP (2000) Mutations in the tuberous sclerosis complex 48. Lochner A, Moolman JA (2006) The many faces of H89: A review. Cardiovasc Drug Rev gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proc Natl 24:261–274. – Acad Sci USA 97:6085 6090. 49. Murray AJ (2008) Pharmacological PKA inhibition: All may not be what it seems. Sci 15. Yu J, Astrinidis A, Henske EP (2001) Chromosome 16 loss of heterozygosity in tu- Signal 1:re4. berous sclerosis and sporadic lymphangiomyomatosis. Am J Respir Crit Care Med 164: 50. Uhler MD, et al. (1986) Isolation of cDNA clones coding for the catalytic subunit of 1537–1540. mouse cAMP-dependent protein kinase. Proc Natl Acad Sci USA 83:1300–1304. 16. Crino PB, Nathanson KL, Henske EP (2006) The tuberous sclerosis complex. N Engl J 51. Uhler MD, Chrivia JC, McKnight GS (1986) Evidence for a second isoform of the cat- Med 355:1345–1356. alytic subunit of cAMP-dependent protein kinase. J Biol Chem 261:15360–15363. 17. Dibble CC, et al. (2012) TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream 52. Turnham RE, Scott JD (2016) catalytic subunit isoform PRKACA; of mTORC1. Mol Cell 47:535–546. History, function and physiology. Gene 577:101–108. 18. Jewell JL, Russell RC, Guan KL (2013) signalling upstream of mTOR. Nat 53. Beebe SJ, et al. (1990) Molecular cloning of a tissue-specific protein kinase (C gamma) Rev Mol Cell Biol 14:133–139. from human testis–Representing a third isoform for the catalytic subunit of cAMP- 19. Rosner M, Hanneder M, Siegel N, Valli A, Hengstschläger M (2008) The tuberous dependent protein kinase. Mol Endocrinol 4:465–475. sclerosis gene products hamartin and tuberin are multifunctional proteins with a 54. Skålhegg BS, et al. (2002) Mutation of the Calpha subunit of PKA leads to growth wide spectrum of interacting partners. Mutat Res 658:234–246. retardation and sperm dysfunction. Mol Endocrinol 16:630–639. 20. Peterson TR, et al. (2009) DEPTOR is an mTOR inhibitor frequently overexpressed in 55. Cao Y, et al. (2014) Activating hotspot L205R mutation in PRKACA and adrenal multiple myeloma cells and required for their survival. Cell 137:873–886. Cushing’s syndrome. Science 344:913–917. 21. Guertin DA, Sabatini DM (2007) Defining the role of mTOR in cancer. Cancer Cell 12: 56. Moore CE, Xie J, Gomez E, Herbert TP (2009) Identification of cAMP-dependent kinase 9–22. as a third in vivo ribosomal protein S6 kinase in pancreatic beta-cells. J Mol Biol 389: 22. Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: From growth signal integration to 480–494. cancer, diabetes and . Nat Rev Mol Cell Biol 12:21–35. 57. January B, et al. (1997) Beta2-adrenergic receptor desensitization, internalization, 23. Sengupta S, Peterson TR, Sabatini DM (2010) Regulation of the mTOR complex and phosphorylation in response to full and partial agonists. J Biol Chem 272: – 1 pathway by nutrients, growth factors, and stress. Mol Cell 40:310–322. 23871 23879. 24. Gressner AM, Wool IG (1974) The phosphorylation of liver ribosomal proteins in vivo. 58. Tran TM, et al. (2004) Characterization of agonist stimulation of cAMP-dependent Evidence that only a single small subunit protein (S6) is phosphorylated. J Biol Chem protein kinase and G protein-coupled receptor kinase phosphorylation of the beta2- 249:6917–6925. adrenergic receptor using phosphoserine-specific antibodies. Mol Pharmacol 65: – 25. Sarbassov DD, et al. (2004) Rictor, a novel binding partner of mTOR, defines a 196 206. rapamycin-insensitive and raptor-independent pathway that regulates the cytoskel- 59. Shenoy SK, McDonald PH, Kohout TA, Lefkowitz RJ (2001) Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science eton. Curr Biol 14:1296–1302. 294:1307–1313. 26. Laplante M, Sabatini DM (2009) mTOR signaling at a glance. J Cell Sci 122:3589–3594. 60. Sette C, Conti M (1996) Phosphorylation and activation of a cAMP-specific phospho- 27. Heitman J, Movva NR, Hall MN (1991) Targets for cell cycle arrest by the immuno- diesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the suppressant rapamycin in yeast. Science 253:905–909. activation. J Biol Chem 271:16526–16534. 28. Huang S, Bjornsti MA, Houghton PJ (2003) Rapamycins: Mechanism of action and 61. Sette C, Iona S, Conti M (1994) The short-term activation of a rolipram-sensitive, cellular resistance. Cancer Biol Ther 2:222–232. cAMP-specific phosphodiesterase by thyroid-stimulating hormone in thyroid FRTL- 29. McCormack FX, et al.; National Institutes of Health Rare Lung Diseases Consortium; 5 cells is mediated by a cAMP-dependent phosphorylation. J Biol Chem 269: MILES Trial Group (2011) Efficacy and safety of sirolimus in lymphangioleiomyoma- 9245–9252. tosis. N Engl J Med 364:1595–1606. 62. Omori K, Kotera J (2007) Overview of PDEs and their regulation. Circ Res 100:309–327. 30. Young L, et al.; MILES Trial Group (2013) Serum VEGF-D a concentration as a bio- 63. Gettys TW, Blackmore PF, Redmon JB, Beebe SJ, Corbin JD (1987) Short-term feedback marker of lymphangioleiomyomatosis severity and treatment response: A prospective regulation of cAMP by accelerated degradation in rat tissues. J Biol Chem 262: analysis of the Multicenter International Lymphangioleiomyomatosis Efficacy of Si- 333–339. – rolimus (MILES) trial. Lancet Respir Med 1:445 452. 64. Oki N, Takahashi SI, Hidaka H, Conti M (2000) Short term feedback regulation of 31. Bissler JJ, et al. (2008) Sirolimus for angiomyolipoma in tuberous sclerosis complex or cAMP in FRTL-5 thyroid cells. Role of PDE4D3 phosphodiesterase activation. J Biol – lymphangioleiomyomatosis. N Engl J Med 358:140 151. Chem 275:10831–10837. 32. Taveira-DaSilva AM, Hathaway O, Stylianou M, Moss J (2011) Changes in lung func- 65. Barnes PJ (2004) Distribution of receptor targets in the lung. Proc Am Thorac Soc 1: tion and chylous effusions in patients with lymphangioleiomyomatosis treated with 345–351. sirolimus. Ann Intern Med 154:797–805. 66. Johnston SL, Edwards MR (2009) Mechanisms of adverse effects of beta-agonists in 33. Krizanova O, Babula P, Pacak K (2016) Stress, catecholaminergic system and cancer. asthma. Thorax 64:739–741. Stress 19:419–428. 67. To Y, et al. (2010) Targeting phosphoinositide-3-kinase-delta with theophylline re- 34. Le CP, et al. (2016) Chronic stress in mice remodels lymph vasculature to promote verses corticosteroid insensitivity in chronic obstructive pulmonary disease. Am J tumour cell dissemination. Nat Commun 7:10634. Respir Crit Care Med 182:897–904. 35. Taveira-DaSilva AM, et al. (2001) Reversible airflow obstruction, proliferation of ab- 68. Mitani A, Ito K, Vuppusetty C, Barnes PJ, Mercado N (2016) Restoration of cortico- normal smooth muscle cells, and impairment of gas exchange as predictors of out- steroid sensitivity in chronic obstructive pulmonary disease by inhibition of mam- come in lymphangioleiomyomatosis. Am J Respir Crit Care Med 164:1072–1076. malian target of rapamycin. Am J Respir Crit Care Med 193:143–153.

10 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1719960115 Le et al. Downloaded by guest on September 26, 2021