Kisspeptins Inhibit Human Airway Smooth Muscle Proliferation

28 Sex/gender disparity in asthma is recognized, and suggests a modulatory role for sex-29 steroids, particularly estrogen. However, studies including our own show a dichotomous role for 30 estrogen in airway remodeling, making it unclear whether sex hormones are protective or 31 detrimental in asthma, and suggesting a need to explore mechanisms upstream or independent 32 of estrogen. We hypothesize that Kisspeptin (Kp)/KISS1R signaling serves this role. Airway 33 smooth muscle (ASM) is a key structural cell type that contributes to remodeling in asthma. We 34 explored the role of Kp/KISS1R in regulating ASM proliferation. We report novel data that Kp and 35 KISS1R are expressed in human airways, especially ASM, with lower expression in ASM from 36 females compared to males, and asthmatics showing lowest expression compared to non-37 asthmatics. Proliferation studies showed that cleaved forms of Kp, particularly Kp-10 mitigates 38 PDGF-induced ASM proliferation. Pharmacological inhibition and shRNA knockdown of KISS1R 39 increased basal ASM proliferation, further amplified by PDGF. The anti-proliferative effect of Kp-40 10 in ASM was found to be mediated by inhibition of MAPK-ERK-Akt pathways, with altered 41 expression of PCNA, C/EBP-alpha, Ki-67, Cyclin-D1, and Cyclin-E leading to cell-cycle arrest at 42 G0/G1 phase. Overall, we demonstrate the importance of Kp/KISS1R signaling in regulating ASM 43 proliferation and a potentially novel therapeutic avenue to blunt remodeling in asthma. 44 45 46 47 48 49 50

Amongst the multiple cell types involved, the airway smooth muscle (ASM) is an important structural cell well-known for contributing to the contractile and AHR aspect (11)(12)(13)(14)(15)(16).However, increased ASM mass is a commonly found pathological feature of asthma, suggesting a role for enhanced ASM proliferation (13,(17)(18)(19), which may further contribute to the increased extracellular matrix (ECM) production within asthmatic airways.Thus, factors that modulate ASM structure and function become important towards understanding asthma pathophysiology for developing novel therapeutic strategies.Here, mechanisms that influence airway remodeling are particularly appealing given that current therapies including corticosteroids are not effective in targeting remodeling in asthma (6).
Asthma shows age-and sex-related differences in epidemiology and clinical manifestation, where prepubertal boys are more likely to have asthma compared to girls, but following puberty, adult women show greater incidence, frequency, and severity of asthma compared to men, a difference that decreases following menopause (1)(2)(3)(4)7,20).Some female asthmatics show exacerbated asthma symptoms during premenstrual or menstrual phases (21,22).These clinical data suggest a functional role for sex-steroids in asthma, especially estrogen.However, the role of estrogen per se in asthma appears paradoxical as some studies suggest that estrogens enhance inflammation (23), while others associate estrogens with an asthma-mitigating role (24).Specific to ASM, we and others have found that differential effects of 17 beta-Estradiol (E2) depend on the contributions of estrogen receptor subtypes (ER-alpha vs. ER-beta) in regulating ASM structure and function (8,9,13,14,16,25,26), thus adding to the complexity of sex steroid signaling in asthma pathophysiology.Furthermore, estrogen fluctuations in perimenopausal females suggest a role for other pathways during the luteal period (27).This brings up a possibility for other mechanisms upstream to or independent of estrogen to play a role in the observed sex differences in asthma.Several pieces of evidence in the central nervous system point to kisspeptin (Kp) being a potential modulatory mechanism upstream of sex steroids: A) Kp is critical for initiating puberty and regulating ovulation via controlling the hypothalamicpituitary-gonadotropic axis (28); B) Kp regulates gonadotropin-releasing hormone and gonadal steroids (29); C) administration of kisspeptin receptor (KISS1R) agonist has been investigated as a potential treatment for sex steroid-dependent diseases (30).Accordingly, Kp/KISS1R's in the airways may be important to understand towards identifying novel pathways that contribute to asthma per se, and potentially the noted sex differences.
Kp is encoded by the KISS1 gene on chromosome 1 that generates a 145 amino acid Kp, which is further cleaved to produce multiple peptides (31) including Kp-10, Kp-13, Kp-14, and Kp-54 which share a common functional C-terminal 10-amino acid sequence (32).Kp serves as an endogenous ligand for KISS1R (GPR54/AXOR12/hOT7T175) (33), a member of the rhodopsin family of GPCRs with sequence homologies to the galanin receptor family (31).Studies in endocrinology and oncology suggest a crucial role for Kp/KISS1R signaling in different cell types (34) with a pivotal contribution to the onset of puberty (28,35) and suppression of cancer metastasis via inhibition of proliferation (36).Kp/KISS1R influences p38 mitogen-associated protein kinases (MAPK) signaling pathways, and can thus modulate inflammation and proliferation (37), two aspects also important for ASM in asthma.However, to the best of our knowledge, there is no information on Kp/KISS1R in the lung, but data in other systems raise the question of whether Kp/KISS1R can regulate airway remodeling in the context of asthma.We hypothesize that Kp/KISS1R signaling has a protective role in regulating ASM mediated airway remodeling: an effect lost during inflammation and/or asthma.To address our hypothesis, the present study was designed to determine 1) the expression of Kp and KISS1R in human ASM; 2) Kp/KISS1R expression levels with respect to sex and asthma; 3) the role of Kp and KISS1R signaling in regulating ASM proliferation and 5) underlying mechanisms of Kp/KISS1R signaling.

Expression of Kp and KISS1R in human lung tissue and ASM cells
Immunohistochemistry of lung tissue sections revealed that both Kp and its receptor KISS1R are expressed in human airways, especially in ASM as determined by colocalization using alpha-smooth muscle actin (alpha-SMA) in lung sections (Figure 1A and B), and by multicolor immunofluorescence staining of isolated human ASM cells (Figure 2A and B).

Sex differences in ASM expression of Kp and KISS1R
Baseline mRNA and protein expression of KISS1 (mRNA)/Kp (protein) and KISS1R in primary non-asthmatic human ASM cells from male vs. female donors showed both KISS1 (P<0.01; Figure 3A)/Kp (P<0.001; Figure 3C) and KISS1R were significantly lower in females compared to males for both mRNA (P<0.01; Figure 3B) and protein (P<0.001; Figure 3D).

Kp and KISS1R in asthmatic ASM
mRNA expression of KISS1 was significantly lower in asthmatic ASM from both males (P<0.01) and females (P<0.05)compared to non-asthmatic counterparts (Figure 4A).This was confirmed by Western analysis for Kp protein expression (P<0.001; Figure 4C).Significant difference was noted for KISS1R mRNA in asthmatic ASM from males and females (P<0.001; Figure 4B) compared to non-asthmatics, again confirmed by analysis for KISS1R protein (P<0.001 for males and P<0.01 for females, Figure 4D).

Cleaved Kps and ASM proliferation
Previous studies have shown that all cleaved forms of Kps show comparable KISS1R receptor binding affinity, but have different downstream potency in terms of cellular effect (38).
Thus, Kp effects may be cell-and context-specific.In order to determine which of the cleaved Kps may be involved in regulating ASM proliferation, we evaluated the effects of 1µM Kp-10, Kp-13 Kp-14, and Kp-54 on basal and PDGF-stimulated proliferation of non-asthmatic ASM cells using MTT assay (Figure 5A).At baseline (without PDGF), Kp-10, Kp-14, and Kp-54 did not significantly alter ASM proliferation, but Kp-13 showed a small but significant (P<0.01)increase compared to vehicle.PDGF substantially increased ASM proliferation (P<0.001) compared to vehicle.However, PDGF's effect on proliferation was significantly blunted by treatment with Kp-10, Kp-14, or Kp-54 (P<0.001), with a lesser effect of Kp-13 (P<0.05) compared to PDGF alone.
Based on these data, we selected Kp-10 as the cleaved form of Kp to further explore.We first performed a concentration dependence study at 100nM, 1µM, and 10µM for PDGF-induced human ASM proliferation using non-asthmatic human ASM cells and found 1µM and 10µM Kp-10 significantly (P<0.001)inhibited proliferation (Figure 5B).Accordingly, we selected 1 µM Kp-10 for subsequent studies.To verify the lack of cytotoxicity from Kp-10, we performed an LDH assay (Figure 5C) with serum-free media as a negative control and 0.1 % Triton-X 100 as positive control and found no cytotoxicity with any of the treatment groups.

Kp-10 effects on proliferation
We performed Brightfield (BF) and MTT assays in both non-asthmatic and asthmatic ASM cells with/without PDGF (Figure 6A and B).In addition, we incorporated a KISS1R inhibitor (KI, Kp-234 trifluoroacetate; 100 nM) as a treatment group to confirm the role of this receptor.As expected, we observed significantly higher proliferation in asthmatic ASM cells compared to nonasthmatics (P<0.05).At baseline, Kp-10 did not significantly alter proliferation in either nonasthmatic or asthmatic ASM.Interestingly, inhibition of KISS1R with KI significantly increased in the basal proliferation of non-asthmatic (P<0.01 in BF assay and P<0.05 in MTT assay) and asthmatic (P<0.01 in BF assay and P<0.05 in MTT assay) ASM cells.Further, ASM cells exposed to PDGF showed a significant (p<0.001 for both BF and MTT assays) increase in proliferation for both non-asthmatic and asthmatic cells with a greater effect on asthmatic ASM (P<0.05 in BF assay and P<0.001 in MTT assay).Pretreatment with Kp-10 significantly blunted the pro-proliferative effect of PDGF in both non-asthmatic (P<0.001) and asthmatic (P<0.001)ASM cells.
Interestingly, PDGF enhancement of proliferation was unaltered by pretreatment with KI.
Similarly, ASM cells first treated with KI and then exposed to Kp-10 did not influence PDGF enhancement of proliferation in either non-asthmatic or asthmatic cells, overall highlighting the importance of KISS1R in mediating Kp-10 effects.
To further confirm the role of KISS1R in Kp-10 effect, we used KISS1R shRNA knockdown (with a scrambled negative shRNA as control; Figure 6C and D).The efficiency of shRNA transduction was confirmed by Western analysis (P<0.001; Figure 6E).Kp-10 alone did not significantly alter basal cell proliferation in both negative and KISS1R knockdown cells.PDGF significantly increased ASM proliferation in negative shRNA transduced non-asthmatic and asthmatic cells (P<0.001 for both BF and MTT assays): effects that were amplified in KISS1R knockdown non-asthmatic and asthmatic ASM cells (P<0.001 both BF and MTT assays).
Pretreatment with Kp-10 significantly lowered PDGF-induced ASM proliferation in negative shRNA transduced non-asthmatic and asthmatic ASM cells (P<0.001 for both BF and MTT assays), but not in KISS1R shRNA knockdown cells.
The above data suggested an autocrine effect of ASM-generated Kp-10 on proliferation.
To confirm an autocrine Kp effect, we measured and found endogenous ASM Kp secretion in the conditional media of both non-asthmatic and asthmatic ASM samples (ELISA; Figure 6F), with significantly lower secretion in asthmatics (P<0.05).

Effect of Kp-10 on markers of proliferation
Cell cycle analysis was performed by flow cytometry using propidium iodide (PI) stained human ASM cells.PDGF significantly increased the number of cells in the 'S' phase compared to vehicle in both non-asthmatic (P<0.05) and asthmatic ASM (P<0.01, Figure 7B), consistent with an increase in proliferation.This mitogenic effect of PDGF was significantly inhibited by Kp-10 pretreatment in both non-asthmatic (P<0.05) and asthmatic (P<0.01)ASM cells.Furthermore, pretreatment with Kp-10 significantly downregulated PDGF induced cell entry into the G2/M phase in both non-asthmatic and asthmatic ASM (P<0.01, Figure 7C).Furthermore, Kp-10 pretreatment increased the numbers of cells in G0/G1 phase compared to PDGF in both non-asthmatic and asthmatic ASM cells (P<0.01, Figure 7A), suggesting the arrest of cell cycle progression in the G0/G1 phase, and again consistent with inhibition of proliferation (Figure 6).
We evaluated the expression of proliferative markers, PCNA and C/EBP-alpha using qRT-PCR and Western analyses, as well as changes in Ki-67 expression by qRT-PCR and nuclear localization by immunofluorescence analysis.PDGF significantly increased the mRNA expression of PCNA (P<0.01),C/EBP-alpha (P<0.01 for non-asthmatics and P<0.001 for asthmatics), and Ki-67 (P<0.001 for non-asthmatics and asthmatics; Figure 8A, B and E).Kp-10 treatment inhibited PDGF-induced increase in mRNA expression of PCNA (P<0.05 for non-asthmatics and P<0.01 for asthmatics), C/EBP-alpha (P<0.01 for non-asthmatics and P<0.05 for asthmatics), and Ki-67 (P<0.001).Consistently, PDGF significantly upregulated protein expression for PCNA (P<0.01 for non-asthmatics and P<0.05 for asthmatics, Figure 8C) and C/EBP-alpha (P<0.001 for nonasthmatics and P<0.05 for asthmatics, Figure 8D).Nuclear localization of Ki-67 was significantly increased with PDGF (P<0.001, Figure 8F), which was further inhibited by Kp-10 (P<0.05 for nonasthmatics and P<0.001 for asthmatics).
PDGF also significantly increased mRNA expression of the cell cycle proteins Cyclin-D1 (P<0.05) and Cyclin E (P<0.001 for non-asthmatics and P<0.01 for asthmatics; Figure 9A and B): effects significantly inhibited in both non-asthmatic (P<0.05) and asthmatic ASM (P<0.01 for Cyclin-D1 and P<0.05 for Cyclin E) pretreated with Kp-10.Consistent results for Cyclin D1 and Cyclin E proteins were observed (Figure 9C and D).PDGF effects on the cell cycle proteins were significantly blunted with Kp-10 pretreatment in both non-asthmatic (P<0.001 for Cyclin-D1 and P<0.01 for Cyclin E) and asthmatic (P<0.05 for both Cyclin-D1 and Cyclin E) ASM cells.
A majority of information regarding Kp derives from studies in the central nervous system, where Kp regulates puberty and hormonal function (28,35).Earlier studies using rat receptors suggested that all the cleaved Kps (Kp-10, Kp-13, Kp-14, and Kp-54) possess similar affinity and efficacy in activating the KISS1R (32).However, data from endocrinology and oncology suggest that the differential effects of Kp fragments are a major aspect of understanding Kp biology (52).
In this regard, while much attention has been given to metastin (a term used interchangeably with Kp), our data suggests that Kp-10 is a potent Kp in the context of ASM and remodeling.
Interestingly, other Kp fragments were less effective in modulating PDGF-induced proliferation.
While other Kp fragments were effective, our data clearly highlight the greater potency of Kp-10, justifying further exploration of this fragment.
There is currently no information on the expression of Kp or KISS1R in the airways.Our novel findings show that Kp and its receptor KISS1R are expressed in human lung tissue, especially in ASM, suggesting a plausible role for Kp/KISS1R signaling in airway biology.Interestingly, we observed lower expression of Kp and KISS1R in ASM from asthmatics, suggesting possible reduced or loss of intrinsic Kp/KISS1R signaling in asthma.Here, increasing evidence from other cell types shows a crucial regulatory role for Kp/KISS1R signaling in cell proliferation and migration by inhibiting NFκB and MAPK signaling pathways (53)(54)(55), which are also relevant to ASM remodeling.The observed reduction of Kp/KISS1R expression and signaling in asthmatics aligns with the increased ASM proliferation observed in asthmatic airways.
In cell proliferation studies, Kp-10, Kp-14 and Kp-54 showed no significant mitogenic effect at baseline per se, suggesting that any modulatory effect of Kps occurs in the presence of extrinsic mitogenic stimuli, as would occur in inflammation or asthma.To stimulate proliferation in ASM cells, we used PDGF as a mitogenic agent given its well-known effects in human ASM (13,(56)(57)(58).PDGF and its receptor (PDGF-R) have been established to play significant roles in airway proliferation and remodeling (56,57,59).Additionally, in earlier studies, we have established that PDGF at a concentration of 2ng/mL significantly induces ASM proliferation via activation of intracellular signaling pathways such as ERK1/2, p38, and Akt (13) that are relevant to both asthma and Kp biology.The increased proliferation observed with PDGF is consistent with our earlier studies ( 13).
An interesting observation was that Kp-10 effects on proliferation were similar between non-asthmatic and asthmatic ASM despite differences in KISS1R expression.This could be due to an enhanced inhibitory effect of Kp-10 via KISS1R in highly proliferative ASM cells or differential signaling of asthmatic ASM.Regardless, what is novel and potentially relevant is the data showing that ASM from both asthmatics and non-asthmatics secretes Kp with autocrine effects on proliferation.Here, KISS1R inhibition by KI significantly increased baseline ASM proliferation suggesting activation of KISS1R by an autocrine mechanism.KI prevented PDGF from further increasing ASM proliferation, which might be due to a ceiling effect.Kp-10 did not reduce PDGFinduced ASM proliferation in KISS1R shRNA knockdown cells, indicating KISS1R dependent activation.Kp-10 via KISS1R activation downregulated PDGF induced expression of Cyclin-D1 and Cyclin-E, thereby initiating cell cycle arrest at G0/G1 phase, consistent with reports from other cell types, which find that Kps initiate cell cycle arrest via KISS1R/GPR54 dependent mechanisms (60).
PCNA and C/EBP-alpha are established markers for proliferation as they play a crucial role during the DNA synthesis phase of mitosis (61,62).PDGF upregulated the expression of PCNA and C/EBP-alpha in ASM cells, which is prevented by Kp-10 pretreatment, further supporting the modulatory role of Kp/KISS1R signaling in ASM proliferation.Notably, previous studies reported a decreased C/EBP-alpha expression in asthma (63,64), whereas our observed data shows increased C/EBP-alpha expression with PDGF exposure in ASM cells from both nonasthmatics and asthmatics.The reason for this discrepancy is unclear and warrants further exploration.Ki-67 is a crucial marker for mitosis as it is exclusively detected in the nucleus during the G1, S, and G2 phases of the cell cycle and absent in the G0 phase (65).PDGF-induced nuclear translocation of Ki-67 in the nucleus was blunted by Kp-10 pre-exposure, further strengthening our findings from ASM cell cycle analysis.Further, the importance of MAPK/ERK signaling pathways in cell growth, proliferation, and differentiation of human ASM cells is well established (13,(66)(67)(68).In other cell systems, KISS1R activation is known to inhibit the phosphorylation of MAPK signaling via beta-arrestin-1 (69).Additionally, KISS1R via regulating alpha sub-unit of Gq/11 inhibits the phosphorylation of PI3K/Akt and subsequently regulates cell growth (69,70).Similarly, our findings reported increased phosphorylation of ERK1/2 and p38 MAPK upon PDGF exposure, which was inhibited by Kp-10 pretreatment.Further, PDGF exposure upregulated the PI3K/Akt signaling which activates mTOR signaling, which functions as a serine/threonine-protein kinase that regulates ASM cell proliferation (71)(72)(73).These effects of Kp-10 via KISS1R activation are consistent with data from other cells types (53,74).The inhibition of Akt phosphorylation by Kp-10 further indicated the importance of Kp/KISS1R regulation of ASM proliferative pathways.
The relevance of Kp/KISS1R expression and signaling in airways further lies in the known sex differences of asthma (1,3,4,75).While there has been much exploration of sex steroid effects, particularly estrogens and their effects on ASM proliferation (13), intracellular calcium ([Ca 2+ ]i) handling (16,76,77), ECM dynamics (14), and cell migration (43), it remains unclear whether female sex steroids are protective or detrimental in asthma, given clinical observations of catamenial asthma, and some lab studies showing estrogens enhance inflammation (23,78), but others associating estrogens with an asthma-mitigating role (13,24,(79)(80)(81)(82)(83)(84).Accordingly, it becomes important to consider if mechanisms upstream or even independent of sex hormones play a role, or alternatively, if asthma involves in loss of intrinsic protective mechanisms.Here, we do find sex differences in Kp and KISS1R expression in human ASM, particularly in asthmatics.How such differences contribute to the sex differences in airway structure/function, or change in asthma, particularly in the concurrent presence of sex steroids, remains to be explored.
Overall, our findings suggest that Kp/KISS1R is abundantly expressed in the human airways, but is lower in asthmatic human ASM.KISS1R activation plays a protective role in regulating ASM proliferation, which is lost during asthma, thereby potentially permitting exacerbated ASM remodeling.This proliferation modulatory effect of cleaved Kp (Kp-10) in ASM is via KISS1R mediated inhibition of p38 MAPK/ERK/Akt signaling pathways, thereby limiting the transcription of PCNA, C/EBP-alpha, Ki-67, Cyclin-D1, and Cyclin-E.Although not the primary focus of this study, we show for the first time the differential expression of Kp/KISS1R in females as compared to males, which may underlie intrinsic differences and endogenous effects in vitro.
We, therefore, lay the foundation for further exploratory studies on Kp/KISS1R signaling in the airways.

Tissue and ASM cells
The procedure for acquiring human lung samples and isolating primary human ASM cells has been described previously (85,86).Formalin-fixed, paraffin-embedded human lung tissue sections were used for immunofluorescence studies.Airway samples denuded of epithelium and ASM tissue were enzymatically dissociated as per the manufacturer's instructions (Worthington Biochemical, Lakewood, NJ, USA) to generate ASM cells.For cells, cultures (<5th passage) were maintained under standard conditions of 37°C (5% CO2, 95% air) using DMEM/F12 supplemented with 10% FBS and 1% antibiotic-antimycotic.

Immunofluorescence studies
Standard immunofluorescence techniques were applied to 6 m thick human lung sections.Briefly, sections were baked (56 o C for 2 h), deparaffinized using xylene and ethanol.Sodium citrate buffer (pH 6.0) was used for antigen retrieval by steaming and further rehydrated in Millipore water.Sections were then permeabilized with 0.1% Triton X-100 in phosphatebuffered saline (1XPBS), blocked with 10% goat serum, and incubated with antibodies against Kp, KISS1R, and alpha-SMA.AlexaFluor-488 for alpha-SMA and AlexaFluor-647 for Kp and KISS1R were used as secondary antibodies with DAPI-AF408 counterstaining for nuclei.Highresolution Z-stack images were captured on a confocal microscope (Zeiss-LSM900 with Airyscan2) (87).
For immunofluorescence detection in human ASM cells, cells were fixed with 4% paraformaldehyde in 1XPBS pH 7.2, washed twice with PBS, and permeabilized using 0.05% Triton X-100 in PBS (permeabilization step was omitted for KISS1R), washed twice with PBS, and blocked with 10% goat serum.Followed by blocking, cells were incubated overnight at 4°C with polyclonal rabbit anti-KISS1R and monoclonal mouse anti-Kp antibodies in different wells.
Primary antibodies were detected with AlexaFluor-647 secondary antibodies for Kp and KISS1R, using phalloidin as a smooth muscle marker with DAPI-AF408 counterstaining for nuclei.Images were acquired with the Zeiss confocal microscope (88,89).

Cell treatments
Human ASM cells grown to confluence in T-75 flasks were trypsinized and mixed in 10% FBS containing medium, counted, and seeded into 100mm culture petri plates (for RNA, Protein, and flow cytometry) or 96 well plates (~7000 cells/well, for proliferation studies).Cells were allowed to adhere overnight and washed twice with PBS.Post-washing, serum medium was replaced with serum-free medium for 24 h to synchronize cell growth.1% serum medium was used as a vehicle to maintain the quiescent phase for the proliferation study.Initial studies showed that among the Kp fragments, Kp-10 was the most effective in modulating ASM proliferation, accordingly subsequent studies focused on this fragment.Efficacy and ASM toxicity of Kp-10 was determined using 3-log concentrations (0.1µM, 1µM, and 10µM), alone and in the presence of PDGF (2ng/mL) (13,14).ASM cells were treated with different concentrations of Kp-10 or KI (90,91) to obtain initial optimal concentration and all subsequent experiments followed a single concentration of Kp-10 or KI.The cytotoxicity of Kp-10 was measured by LDH-Cytotoxicity Colorimetric Assay Kit as per the manufacturer's protocol.To measure the endogenous ASM Kp secretion, the media were collected after 48 h serum deprivation and concentrated equally to 500 µL using Amicon Ultra-15 Centrifugal Filter Units (Cat# UFC901024).The endogenous Kp levels in the concentrated conditional media was measured using the Kp ELISA kit as per the manufacturer's protocol.Human ASM cells were exposed to Kp-10 or KI in the presence/absence of PDGF, added after 2 h of pre-incubation with respective treatment groups, and incubated for a total time of 6 h (RNA) and 24 h (proteins) (13).

shRNA Lentiviral Particle Transduction
Non-asthmatic and asthmatic human ASM cells were cultured in 6-well plates to approximately 50-60% confluence.Transfection was achieved using a 20 µl viral stock containing 1X10 5 infectious units of the virus (IFU) for control and KISS1R/GPR54 shRNA lentiviral particles.
Once cells reached 60-70%, serum medium was replaced with fresh 5% serum medium (no antibiotics) and with polybrene (Santa Cruz Cat# sc-134220).Lentiviral particles were thawed, added and the cells incubated overnight.The medium was replaced with a 5% serum medium containing 1% AbAm without polybrene and further incubated for 24 h.After ensuring growth, the medium was replaced with a 10% serum medium with antibiotics for 48 h.For the selection of cells stably transfected with shRNA, the medium was replaced with a 10% serum medium containing 5µg/mL puromycin and incubated for 48-72 h.Cells were replenished with medium every 3-4 days until several puromycin-resistant colonies were identified.Once colonies reached 50% confluence, cells were expanded by transferring them into T-25 or T-75 cell culture flasks.
Efficacy and successful shRNA transduction were verified by Western analysis.

Brightfield Cell Count
After 24 h of respective treatments, total numbers of cells were counted in each of the 96well plates using a high-contrast, bright-field direct cell counting on a Lionheart FX Automated Microscope (LFX; BioTek Instruments, Winooski, VT, USA) (13).

MTT Cell Proliferation Assay
Following cell counting, the medium was aspirated carefully from the wells and replaced with 100 µL of 1% serum medium.For each well 10 µL MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, 5 mg/mL) reagent was then added and incubated at 37˚C for 4 h.The medium was aspirated followed by the addition of 100 µL dimethylsulfoxide (DMSO), and cells were maintained with gentle shaking for 15 min at room temperature.Absorbance at 570 nm was then measured using a Synergy HTX Multi-Mode Plate Reader (BioTek Instruments, Winooski, VT, USA).

Flow cytometry
12-well culture plates were seeded with non-asthmatic and asthmatic ASM cells and treated with appropriate treatments for 24 h.Cells were trypsinized and fixed with ice-cold ethanol followed by centrifugation at 2,000g.Pellets were washed and resuspended in PBS followed by propidium iodide (PI; 3 µM) staining for 15 min.PI-stained cells were analyzed using BD Accuri® C6 Flow cytometer (BD Biosciences, San Jose, CA USA).A minimum of 40,000 events were captured per sample and cells were analyzed for G0/G1, S, and G2/M phase using BD Accuri™ C6 Plus software (13).

Western Blot Analysis
Human ASM cell lysates for individual treatment groups were prepared using cell lysis buffer as previously described (25).Briefly, cells were washed once with PBS and thereafter vortexed in lysis buffer supplemented with protease and phosphatase inhibitors, and subsequent supernatants were determined for protein content using a DC Protein Assay kit (BioRad, Hercules, CA, USA).30 µg equivalent protein from respective treatment groups were loaded on 4-15% gradient gels (Criterion Gel System; Bio-Rad) and transferred onto 0.22 µm PVDF membranes using a Bio-Rad Trans-Blot Turbo rapid transfer system.5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h at room temperature was used as a blocking buffer and membranes were incubated overnight at 4°C with specific primary antibodies of interest.
Following three washes for 8 min/wash with Tris-buffered saline containing 0.1% tween (TBST), blots were incubated with LiCOR near-red conjugated secondary antibodies at room temperature for 1 h.Beta-actin was used as a loading control.Protein expression was determined by imaging the membrane on a Li-Cor Odyssey XL system and densitometry analysis was performed using Image Studio Lite software.Western blot analysis was performed by normalizing the raw values of the protein of interest to respective raw values of beta-actin.

qRT-PCR analysis
Cells were washed with RNA-grade PBS before trypsinization and proceeded for RNA isolation.RNA cell isolation was performed using the Quick-RNA™ MiniPrep kit (Zymo Research, Irvine, CA, USA) and OneScript cDNA Synthesis Kit (ABM biological materials, Richmond, BC, Canada) were used for complementary DNA (cDNA) synthesis using a minimum of 500 ng of RNA for each sample.Genomic DNA contamination was avoided by using DNAse I treatment.

Statistical Analysis
Human ASM cells from at least five donors (males and females; non-asthmatics and asthmatics) were used for all experiments.For expression studies, cell lysates for Western analysis and cDNA for qRT-PCR were obtained from at least five different individual donor samples.Statistical analysis was performed using a one-tailed unpaired t-test or one-way/twoway ANOVA followed by Tukey's post-hoc multiple comparisons test using GraphPad Prism version 9.1.0for Windows.Statistical significance was tested at a minimum of p<0.05 level.All values are expressed as mean ± SEM.

Study Approval
Human bronchi from third-to sixth generations were isolated from lung specimen's incidental to donor thoracic surgeries at Mayo Clinic (focal, noninfectious indications; typically, lobectomies, rarely pneumonectomies).Normal lung areas were identified by a pathologist.Donors with no prior history of obstructive lung disease (including COPD) were considered to have an otherwise normal lung function and classified as non-asthmatics (de-identified pulmonary conditions of the donors).The protocols were approved by the Mayo Clinic Institutional Review Board (IRB#08-002518).qRT-PCR and Western analysis data showed lower baseline Kp (A, C) and KISS1R (B, D) expression in asthmatic ASM cells compared to non-asthmatics.Interestingly, Kp and KISS1R expression were found to be low in both male and female asthmatic ASM cells compared to nonasthmatic ASM cells.mRNA expression of KISS1 (A) and KISS1R (B) normalized with housekeeping gene s16 and represented as Ct.The protein expression of Kp (C) and KISS1R (D) were determined by Western analysis in human ASM cells and normalized with beta-actin as a loading control.Data are represented as a minimum to maximum of 7-9 individual donor ASM samples from male and female and analyzed using two-way ANOVA followed by Tukey's posthoc test.*P<0.05,**P<0.01,***P<0.001vs. non-asthmatic male; # P<0.05, ## P<0.01, ### P<0.001 vs. non-asthmatic female; $ P<0.05, $$$ P<0.001 vs. asthmatic male.agonist, Kp-10 significantly blunted the mitogenic effect on PDGF in non-asthmatic and asthmatic human ASM cells as evaluated by high contrast brightfield (A) and MTT (B) assays.KISS1Rinhibitor (KI; Kp-234 trifluoroacetate) showed a significant increase in basal ASM proliferation compared to vehicle and did not show any changes in PDGF-induced proliferation in both asthmatic and non-asthmatic ASM cells.Human ASM cells transduced with KISS1R specific shRNA show increased basal ASM proliferation compared to negative shRNA.Further, we did not observe any significant reduction in PDGF-induced proliferation after Kp-10 treatment in KISS1R shRNA transduced cells as measured by high contrast brightfield (C) and MTT (D) assays.Western blot analysis was performed to confirm the transduction efficacy of KISS1R shRNA in non-asthmatic and asthmatic ASM cells (E).The endogenous Kp secretion of ASM was measured by ELISA using conditioned media.The calibration of ELISA was confirmed by a standard curve of Kp (F, top panel).ASM cells from asthmatics showed reduced Kp secretion in conditioned media compared to non-asthmatics (F, bottom panel).Data are represented as a minimum to maximum of 5-7 individual donor ASM samples from non-asthmatic/asthmatic and analyzed using one/two-way ANOVA followed by Tukey's post-hoc test or one-tailed unpaired ttest.*P<0.05,**P<0.01,***P<0.001vs. non-asthmatic vehicle or negative shRNA vehicle; ### P<0.001 vs. respective PDGF-exposed group; $ P<0.05, $$ P<0.01, $$$ P<0.001 vs. respective non-asthmatics or negative shRNA groups.

Figure 1 .
Figure 1.Kisspeptin (Kp) and its receptor, KISS1R are expressed in the human airway as shown by immunohistochemistry. Human lung sections were immunostained for Kp (A, AF-647) and KISS1R (B, AF-647) with alpha-smooth muscle actin (alpha-SMA, AF-488) as a reference marker for smooth muscle-specific colocalization.DAPI was used to stain the nucleus (AF-408).Z-stack images were taken on a Zeiss LSM900 confocal microscope with Airyscan2 settings.Scale bar: 50 µm (insert: 5 µm).Representative images from n = 5 independent nonasthmatic donor samples.

Figure 2 .
Figure 2. Kp and KISS1R expression in isolated human airway smooth muscle (ASM) cells.Paraformaldehyde fixed human ASM cells were immunostained with Kp (A) and KISS1R (B).Kp and KISS1R were probed with AF-647 secondary antibodies and actin filaments with phalloidin (AF-488).DAPI was used to stain the nucleus (AF-408).Z-stack images were taken on a Zeiss LSM900 confocal microscope with Airyscan2 settings.Scale bar: 20 µm.Representative images from n = 5 independent non-asthmatic donor samples.

Figure 3 .
Figure 3. Kp and KISS1R expression in primary human ASM cells from males and females.qRT-PCR and Western analysis data show lower baseline Kp (A, C) and KISS1R (B, D) expression in ASM cells from females compared to males.mRNA expression of KISS1 (A) and KISS1R (B) normalized with housekeeping gene s16 and represented as Ct.Western blots of Kp (C) and KISS1R (D) are representative results from independent experiments.All proteins were normalized to beta-actin.Data are represented as a minimum to maximum of 7-9 individual nonasthmatic male or female donor samples and analyzed using a one-tailed unpaired t-test.**P<0.01,***P<0.001vs. males.

Figure 4 .
Figure 4. Kp and KISS1R expression in non-asthmatic and asthmatic human ASM cells.qRT-PCRand Western analysis data showed lower baseline Kp (A, C) and KISS1R (B, D) expression in asthmatic ASM cells compared to non-asthmatics.Interestingly, Kp and KISS1R expression were found to be low in both male and female asthmatic ASM cells compared to nonasthmatic ASM cells.mRNA expression of KISS1 (A) and KISS1R (B) normalized with housekeeping gene s16 and represented as Ct.The protein expression of Kp (C) and KISS1R (D) were determined by Western analysis in human ASM cells and normalized with beta-actin as a loading control.Data are represented as a minimum to maximum of 7-9 individual donor ASM samples from male and female and analyzed using two-way ANOVA followed by Tukey's posthoc test.*P<0.05,**P<0.01,***P<0.001vs. non-asthmatic male; # P<0.05, ## P<0.01, ### P<0.001 vs. non-asthmatic female; $ P<0.05, $$$ P<0.001 vs. asthmatic male.

Figure 5 .
Figure 5. Kisspeptins and human ASM cell proliferation.The effect of different Kisspeptins (Kp-10, Kp-13 Kp-14, and Kp-54) on basal and PDGF-induced cell proliferation in non-asthmatic human ASM cells was evaluated using MTT assay (A).Amongst the three forms of Kisspeptins (1 µM), Kp-10 significantly blunted PDGF-induced ASM cell proliferation.Further, the effect of different log concentrations of Kp-10 (100nM, 1µM, and 10µM) on regulating PDGF-induced ASM proliferation was determined (B).The relative level of lactate dehydrogenase (LDH) was measured in ASM cell supernatants to evaluate the cytotoxicity of the selected concentration (1 µM) of Kp-10 (C).The treatment groups did not show cytotoxicity after 24 hr of exposure when compared to the negative control group.Data are represented as a minimum to maximum of 7-8 individual non-asthmatic donor ASM samples and analyzed using one-way ANOVA followed by Tukey's post-hoc test.**P<0.01,***P<0.001vs. respective vehicle; # P<0.05, ### P< 0.001 vs. PDGF-exposed groups.

Figure 6 .
Figure 6.KISS1R activation and PDGF-induced proliferation of human ASM cells.KISS1Ragonist, Kp-10 significantly blunted the mitogenic effect on PDGF in non-asthmatic and asthmatic human ASM cells as evaluated by high contrast brightfield (A) and MTT (B) assays.KISS1Rinhibitor (KI; Kp-234 trifluoroacetate) showed a significant increase in basal ASM proliferation compared to vehicle and did not show any changes in PDGF-induced proliferation in both asthmatic and non-asthmatic ASM cells.Human ASM cells transduced with KISS1R specific shRNA show increased basal ASM proliferation compared to negative shRNA.Further, we did not observe any significant reduction in PDGF-induced proliferation after Kp-10 treatment in KISS1R shRNA transduced cells as measured by high contrast brightfield (C) and MTT (D) assays.Western blot analysis was performed to confirm the transduction efficacy of KISS1R shRNA in non-asthmatic and asthmatic ASM cells (E).The endogenous Kp secretion of ASM was measured by ELISA using conditioned media.The calibration of ELISA was confirmed by a standard curve of Kp (F, top panel).ASM cells from asthmatics showed reduced Kp secretion in

Figure 7 .
Figure 7. Effect of Kp-10 on different phases of human ASM cell cycle studied using flow cytometry.Cell cycle analysis showed decreased cell population in G0/G1 phase (A) while increased cell numbers in S and G2/M phases (B and C) with PDGF treatment.Kp-10 treatment significantly reversed the PDGF effect and show reduced cell populations in S and G2/M phases suggesting the arrest of cell cycle progression in the G0/G1 phase.The representative profiles of non-asthmatic (D) and asthmatic (E) human ASM cell cycle distributions by various treatment groups are depicted.Data are represented as a minimum to maximum of 5 individual samples from non-asthmatic/asthmatic donors and analyzed using one-way ANOVA followed by Tukey's post-hoc test.*P<0.05**P<0.01 vs. respective vehicle; # P<0.05, ## P<0.01 vs. PDGF-exposed group.

Figure 8 .
Figure 8.Effect of Kp-10 on PDGF-induced human ASM cell proliferative marker proteins.Non-asthmatic and asthmatic human ASM cells showed increased mRNA and protein expression of PCNA (A, C) and C/EBP-alpha (B, D) following PDGF exposure, an effect substantially blunted by Kp-10.Similarly, PDGF-induced mRNA expression and nuclear localization of Ki67 (E-H) were significantly reduced by Kp-10 treatment.Scale bar: 100 µm.Data are represented as a minimum to maximum of 5-6 individual samples from non-asthmatic/asthmatic donors and analyzed using

Figure 9 .
Figure 9.Effect of Kp-10 on PDGF-induced human ASM cell cycle progression proteins.mRNA and protein expression of Cyclin-D1 (A, C) and Cyclin E (B, D) were significantly increased in non-asthmatic and asthmatic ASM cells upon PDGF exposure.Pretreatment with Kp-10 significantly decreased PDGF-induced mRNA and protein expressions of Cyclin-D1 and Cyclin E. Data are represented as a minimum to maximum of 5-6 individual samples from non-asthmatic/ asthmatic donors and analyzed using one-way ANOVA followed by Tukey's post-hoc test.*P<0.05,**P<0.01,***P<0.001vs. respective vehicle; # P<0.05, ## P<0.01, ### P<0.001 vs. PDGFexposed groups.

Figure 10 .
Figure 10.Effect of Kp-10 on human ASM proliferative signaling pathways.Western analysis showed PDGF-induced activation of p38 (A) Akt (B) and ERK1/2 (C) in human ASM cells.Pretreatment with Kp-10 significantly reduced the PDGF-induced phosphorylation of p38, Akt, and ERK1/2.Data are represented as a minimum to maximum of 6 individual samples from nonasthmatics and analyzed using one-way ANOVA followed by Tukey's post-hoc test.*P<0.05 vs. Vehicle, # P<0.05, ## P<0.01 vs. PDGF-exposed groups.