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We present BVRI and unfiltered light curves of 93 Type Ia supernovae (SNe Ia) from the Lick Observatory Supernova Search (LOSS) follow-up program conducted between 2005 and 2018. Our sample consists of 78 spectroscopically normal SNe Ia, with the remainder divided between distinct subclasses (3 SN 1991bg-like, 3 SN 1991T-like, 4 SNe Iax, 2 peculiar, and 3 super-Chandrasekhar events), and has a median redshift of 0.0192. The SNe in our sample have a median coverage of 16 photometric epochs at a cadence of 5.4 d, and the median first observed epoch is ∼4.6 d before maximum B-band light. We describe how the SNe in our sample are discovered, observed, and processed, and we compare the results from our newly developed automated photometry pipeline to those from the previous processing pipeline used by LOSS. After investigating potential biases, we derive a final systematic uncertainty of 0.03 mag in BVRI for our data set. We perform an analysis of our light curves with particular focus on using template fitting to measure the parameters that are useful in standardizing SNe Ia as distance indicators. All of the data are available to the community, and we encourage future studies to incorporate our light curves in their analyses.

Posture control is based on central integration of multisensory inputs, and on internal representation of body orientation in space. This multisensory feedback regulates posture control and continuously updates the internal model of body’s position which in turn forwards motor commands adapted to the environmental context and constraints. The peripheral localization of the vestibular system, close to the cochlea, makes vestibular damage possible following cochlear implant (CI) surgery. Impaired vestibular function in CI patients, if any, may have a strong impact on posture stability. The simple postural task of quiet standing is generally paired with cognitive activity in most day life conditions, leading therefore to competition for attentional resources in dual-tasking, and increased risk of fall particularly in patients with impaired vestibular function. This study was aimed at evaluating the effects of post-lingual cochlear implantation on posture control in adult deaf patients. Possible impairment of vestibular function was assessed by comparing the postural performance of patients to that of age-matched healthy subjects during a simple postural task performed in static and dynamic conditions, and during dual-tasking with a visual or auditory memory task. Postural tests were done in eyes open (EO) and eyes closed (EC) conditions, with the cochlear implant activated (ON) or not (OFF). Results showed that the CI patients significantly reduced limits of stability and increased postural instability in static conditions. In dynamic conditions, they spent considerably more energy to maintain equilibrium, and their head was stabilized neither in space nor on trunk while the controls showed a whole body rigidification strategy. Hearing (prosthesis on) as well as dual-tasking did not really improve the dynamic postural performance of the CI patients. We conclude that CI patients become strongly visual dependent mainly in challenging postural conditions.

The 2023-2032 Planetary Science and Astrobiology Decadal Survey prioritized the Uranus Orbiter and Probe (UOP) mission concept as the next priority flagship mission. The UOP concept includes scientific studies of the Uranian moon system. Although the Uranian moons differ greatly from the ocean worlds in the Jovian and Saturnian systems, the emerging hypothesis is that some of them could at least sustain thin, potentially concentrated, oceans. Herein, we make a case that these moons are important and interesting targets of astrobiological research. Studying these worlds would provide critical astrobiological data related to their habitability, including origin, evolution, and potential death, as well as the formation and evolution of ocean worlds more broadly. There is a strong need for research that connects astrobiology to modeling and experimentation to better characterize the possible conditions of these worlds, and this will be critical in formulating and maximizing the potential science that could be done by a Uranus flagship mission.

The habitability of Europa is a property within a system, which is driven by a multitude of physical and chemical processes and is defined by many interdependent parameters, so that its full characterization requires collaborative investigation. To explore Europa as an integrated system to yield a complete picture of its habitability, the Europa Clipper mission has three primary science objectives: (1) characterize the ice shell and ocean including their heterogeneity, properties, and the nature of surface-ice-ocean exchange; (2) characterize Europa's composition including any non-ice materials on the surface and in the atmosphere, and any carbon-containing compounds; and (3) characterize Europa's geology including surface features and localities of high science interest. The mission will also address several cross-cutting science topics including the search for any current or recent activity in the form of thermal anomalies and plumes, performing geodetic and radiation measurements, and assessing high-resolution, co-located observations at select sites to provide reconnaissance for a potential future landed mission. Synthesizing the mission's science measurements, as well as incorporating remote observations by Earth-based observatories, the James Webb Space Telescope, and other space-based resources, to constrain Europa's habitability, is a complex task and is guided by the mission's Habitability Assessment Board (HAB)., Competing Interests: Competing InterestsThe authors have no competing interests to declare that are relevant to the content of this article., (© The Author(s) 2023.)

The Galileo mission to Jupiter revealed that Europa is an ocean world. The Galileo magnetometer experiment in particular provided strong evidence for a salty subsurface ocean beneath the ice shell, likely in contact with the rocky core. Within the ice shell and ocean, a number of tectonic and geodynamic processes may operate today or have operated at some point in the past, including solid ice convection, diapirism, subsumption, and interstitial lake formation. The science objectives of the Europa Clipper mission include the characterization of Europa's interior; confirmation of the presence of a subsurface ocean; identification of constraints on the depth to this ocean, and on its salinity and thickness; and determination of processes of material exchange between the surface, ice shell, and ocean. Three broad categories of investigation are planned to interrogate different aspects of the subsurface structure and properties of the ice shell and ocean: magnetic induction, subsurface radar sounding, and tidal deformation. These investigations are supplemented by several auxiliary measurements. Alone, each of these investigations will reveal unique information. Together, the synergy between these investigations will expose the secrets of the Europan interior in unprecedented detail, an essential step in evaluating the habitability of this ocean world., Competing Interests: Competing InterestsThe authors declare that they have no conflicts of interest., (© The Author(s) 2023.)

The mechanisms for chaos terrain formation on Europa have long been a source of debate in the scientific community. There exist numerous theoretical and numerical models for chaos formation, but to date there has been a lack of quantifiable observations that can be used to constrain models and permit comparison to the outputs of these chaos models. Here, we use mapping and statistical analysis to develop a quantitative description of chaos terrain and their observed morphologies. For nine chaos features, we map every block, or region of pre-existing terrain within disrupted matrix. We demonstrate that chaos terrains follow a continuous spectrum of morphologies between two endmembers, platy and knobby. We find that any given chaos terrain's morphology can be quantified by means of the linearized exponential slope of its cumulative block area distribution. This quantitative metric provides a new diagnostic parameter in future studies of chaos terrain formation and comparison., (© 2022 Jet Propulsion Laboratory. California Institute of Technology. Government sponsorship acknowledged.)

Purpose: This phase II study investigated daily or weekly sapanisertib (a selective dual inhibitor of mTOR complexes 1 and 2) in combination with fulvestrant., Patients and Methods: Postmenopausal women with estrogen receptor-positive (ER+)/HER2-negative (HER2-) advanced or metastatic breast cancer following progression during/after aromatase inhibitor treatment were randomized to receive fulvestrant 500 mg (28-day treatment cycles), fulvestrant plus sapanisertib 4 mg daily, or fulvestrant plus sapanisertib 30 mg weekly, until progressive disease, unacceptable toxicity, consent withdrawal, or study completion., Results: Among 141 enrolled patients, baseline characteristics were balanced among treatment arms, including prior cyclin-dependent kinase-4/6 (CDK4/6) inhibitor treatment in 33% to 35% of patients. Median progression-free survival (PFS; primary endpoint) was 3.5 months in the single-agent fulvestrant arm, compared with 7.2 months for fulvestrant plus sapanisertib daily [HR, 0.77; 95% confidence interval (CI), 0.47-1.26] and 5.6 months for fulvestrant plus sapanisertib weekly (HR, 0.88; 95% CI, 0.53-1.45). The greatest PFS benefits were seen in patients who had previously received CDK4/6 inhibitors. The most common adverse events were nausea, vomiting, and hyperglycemia, all occurring more frequently in the combination therapy arms. Treatment discontinuation due to adverse events occurred more frequently in the two combination therapy arms than with single-agent fulvestrant (32% and 36% vs. 4%, respectively)., Conclusions: Fulvestrant plus sapanisertib daily/weekly resulted in numerically longer PFS in patients with ER+/HER2- advanced or metastatic breast cancer, compared with single-agent fulvestrant. The combination was associated with increased toxicity. Further development of sapanisertib using these dosing schedules in this setting is not supported by these data., (©2022 The Authors; Published by the American Association for Cancer Research.)

Purpose: This open-label, multicenter, phase IB/II study evaluated sapanisertib, a dual inhibitor of mTOR kinase complexes 1/2, plus exemestane or fulvestrant in postmenopausal women with hormone receptor-positive (HR + )/HER2-negative (HER2 - ) advanced/metastatic breast cancer., Patients and Methods: Eligible patients had previously progressed on everolimus with exemestane/fulvestrant and received ≤3 (phase IB) or ≤1 (phase II) prior chemotherapy regimens. Patients received sapanisertib 3 to 5 mg every day (phase IB), or 4 mg every day (phase II) with exemestane 25 mg every day or fulvestrant 500 mg monthly in 28-day cycles. Phase II enrolled parallel cohorts based on prior response to everolimus. The primary objective of phase II was to evaluate antitumor activity by clinical benefit rate at 16 weeks (CBR-16)., Results: Overall, 118 patients enrolled in phase IB ( n = 24) and II ( n = 94). Five patients in phase IB experienced dose-limiting toxicities, at sapanisertib doses of 5 mg every day ( n = 4) and 4 mg every day ( n = 1); sapanisertib 4 mg every day was the MTD in combination with exemestane or fulvestrant. In phase II, in everolimus-sensitive versus everolimus-resistant cohorts, CBR-16 was 45% versus 23%, and overall response rate was 8% versus 2%, respectively. The most common adverse events were nausea (52%), fatigue (47%), diarrhea (37%), and hyperglycemia (33%); rash occurred in 17% of patients. Molecular analysis suggested positive association between AKT1 mutation status and best treatment response (complete + partial response; P = 0.0262)., Conclusions: Sapanisertib plus exemestane or fulvestrant was well tolerated and exhibited clinical benefit in postmenopausal women with pretreated everolimus-sensitive or everolimus-resistant breast cancer., (©2021 American Association for Cancer Research.)

Introduction: We assessed the Aurora A kinase inhibitor, alisertib, plus paclitaxel (henceforth referred to as alisertib/paclitaxel) as second-line treatment for SCLC., Methods: In this double-blind study, patients with relapsed or refractory SCLC were stratified by relapse type (sensitive versus resistant or refractory) and brain metastases and randomized 1:1 to alisertib/paclitaxel or placebo plus paclitaxel (henceforth referred to as placebo/paclitaxel) in 28-day cycles. The primary end point was progression-free survival (PFS). Associations of c-Myc expression in tumor tissue (prespecified) and genetic alterations in circulating tumor DNA (retrospective) with clinical outcome were evaluated., Results: A total of 178 patients were enrolled (89 in each arm). The median PFS was 3.32 months with alisertib/paclitaxel versus 2.17 months with placebo/paclitaxel (hazard ratio [HR] = 0.77, 95% confidence limit [CI]: 0.557-1.067, p = 0.113 in the intent-to-treat population versus HR = 0.71, 95% CI: 0.509-0.985, p = 0.038 with corrected analysis applied). Among 140 patients with genetic alternations, patients with cell cycle regulator mutations (cyclin-dependent kinase 6 gene [CDK6], retinoblastoma-like 1 gene [RBL1], retinoblastoma-like 2 gene [RBL2], and retinoblastoma 1 gene [RB1]) had significantly improved PFS with alisertib/paclitaxel versus with placebo/paclitaxel (3.68 versus 1.80 months, respectively [HR = 0.395, 95% CI: 0.239-0.654, p = 0.0003]), and overall survival (7.20 versus 4.47 months, respectively [HR = 0.427, 95% CI: 0.259-0.704, p = 0.00085]). A subset of patients with c-Myc expression showed significantly improved PFS with alisertib/paclitaxel. The incidence of grade 3 or higher drug-related adverse events was 67% (58 patients) with alisertib/paclitaxel versus 22% (25 patients) with placebo/paclitaxel. Twelve patients (14%) versus 11 (12%) died on study, including four versus zero treatment-related deaths., Conclusions: Efficacy signals were seen with alisertib/paclitaxel in relapsed or refractory SCLC. c-Myc expression and mutations in cell cycle regulators may be potential predictive biomarkers of alisertib efficacy; further prospective validations are warranted., (Copyright © 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Purpose: The aim of this open-label, first-in-setting, randomized phase III trial was to evaluate the efficacy of alisertib, an investigational Aurora A kinase inhibitor, in patients with relapsed/refractory peripheral T-cell lymphoma (PTCL)., Patients and Methods: Adult patients with relapsed/refractory PTCL-one or more prior therapy-were randomly assigned 1:1 to receive oral alisertib 50 mg two times per day (days 1 to 7; 21-day cycle) or investigator-selected single-agent comparator, including intravenous pralatrexate 30 mg/m 2 (once per week for 6 weeks; 7-week cycle), or intravenous gemcitabine 1,000 mg/m 2 or intravenous romidepsin 14 mg/m 2 (days 1, 8, and 15; 28-day cycle). Tumor tissue (disease subtype) and imaging were assessed by independent central review. Primary outcomes were overall response rate and progression-free survival (PFS). Two interim analyses and one final analysis were planned., Results: Between May 2012 and October 2014, 271 patients were randomly assigned (alisertib, n = 138; comparator, n = 133). Enrollment was stopped early on the recommendation of the independent data monitoring committee as a result of the low probability of alisertib achieving PFS superiority with full enrollment. Centrally assessed overall response rate was 33% for alisertib and 45% for the comparator arm (odds ratio, 0.60; 95% CI, 0.33 to 1.08). Median PFS was 115 days for alisertib and 104 days for the comparator arm (hazard ratio, 0.87; 95% CI, 0.637 to 1.178). The most common adverse events were anemia (53% of alisertib-treated patients v 34% of comparator-treated patients) and neutropenia (47% v 31%, respectively). A lower percentage of patients who received alisertib (9%) compared with the comparator (14%) experienced events that led to study drug discontinuation. Of 26 on-study deaths, five were considered treatment related (alisertib, n = 3 of 11; comparator, n = 2 of 15). Two-year overall survival was 35% for each arm., Conclusion: In patients with relapsed/refractory PTCL, alisertib was not statistically significantly superior to the comparator arm.

Importance: There is an unmet medical need for the treatment of recurrent ovarian cancer, and new approaches are needed to improve progression-free survival (PFS) and overall survival., Objective: This phase 1/2 study evaluated the activity of alisertib in combination with weekly paclitaxel in patients with breast (phase 1) and ovarian cancer (phase 1 and phase 2)., Design, Setting, and Participants: An open-label phase 1 and randomized phase 2 clinical trial conducted from April 16, 2010, for phase 1 and March 28, 2012, to August 12, 2013, for phase 2 was conducted at 33 sites (United States, France, and Poland). Data are reported from a cutoff date of August 12, 2014, with a median duration of follow-up of 7.2 months in the alisertib plus paclitaxel arm and 4.6 months in the paclitaxel arm. A total of 191 women with advanced breast (phase 1 only) or recurrent ovarian cancer were enrolled, including 142 patients randomized to alisertib plus paclitaxel (n = 73) or paclitaxel alone (n = 69) in the phase 2 study., Interventions: Patients were randomized 1:1 stratified by platinum-free interval (refractory, 0-6 months, 6-12 months) and prior weekly taxane treatment (yes, no) to receive alisertib 40 mg twice per day orally and 3 days on and 4 days off for 3 weeks, plus paclitaxel (60 mg/m2 intravenously, days 1, 8, and 15), or weekly paclitaxel 80 mg/m2 intravenously in 28-day cycles., Main Outcomes and Measures: Primary endpoint was PFS; primary efficacy analysis and safety analysis used modified intention to treat (mITT) population (all randomized patients who received ≥1 dose of study drug)., Results: The median age for the 191 patients enrolled in phase 1 was 59 (range, 29-75) years. The median age for the 142 patients enrolled in phase 2 was 63 (range, 30-81) years for patients receiving alisertib plus paclitaxel and 61 (range, 41-81) years for patients receiving paclitaxel. At data cutoff, 107 (75%) patients had a documented PFS event; 52 (71%) in the alisertib plus paclitaxel arm, and 55 (80%) in the paclitaxel arm. Median PFS was 6.7 months with alisertib plus paclitaxel vs 4.7 months with paclitaxel (HR, 0.75; 80% CI, 0.58-0.96; P = .14; 2-sided P value cutoff = .20 to be considered worthy of further investigation). Drug-related grade 3 or higher adverse events were reported in 63 (86%) vs 14 (20%) patients in the alisertib plus paclitaxel and paclitaxel arms, including 56 (77%) vs 7 (10%) neutropenia, 18 (25%) vs 0 stomatitis, and 10 (14%) vs 2 (3%) anemia; 54 (74%) vs 17 (25%) had adverse events leading to dose reductions. Two patients died during the study (1 in each arm); neither death was considered related to study drug., Conclusions and Relevance: The primary endpoint, PFS, significantly favored alisertib plus paclitaxel over paclitaxel alone. Further investigation is warranted., Trial Registration: ClinicalTrials.gov identifier: NCT01091428.

Purpose: The aurora A kinase inhibitor alisertib demonstrated single-agent clinical activity and preclinical synergy with vincristine/rituximab in B-cell non-Hodgkin lymphoma (B-NHL). This phase I study aimed to determine the safety and recommended phase II dose (RP2D) of alisertib in combination with rituximab ± vincristine in patients with relapsed/refractory aggressive B-NHL., Patients and Methods: Patients with relapsed/refractory, diffuse, large, or other aggressive B-NHL received oral alisertib 50 mg b.i.d. days 1 to 7, plus i.v. rituximab 375 mg/m 2 on day 1, for up to eight 21-day cycles (MR). Patients in subsequent cohorts (3 + 3 design) received increasing doses of alisertib (30 mg starting dose; 10 mg increments) b.i.d. days 1 to 7 plus rituximab and vincristine [1.4 mg/m 2 (maximum 2 mg) days 1, 8] for 8 cycles (MRV). Patients benefiting could continue single-agent alisertib beyond 8 cycles. Cell-of-origin and MYC/BCL2 IHC was performed on available archival tissue., Results: Forty-five patients participated. The alisertib RP2D for MR was 50 mg b.i.d. For MRV ( n = 32), the RP2D was determined as 40 mg b.i.d. [1 dose-limiting toxicity (DLT) at 40 mg; 2 DLTs at 50 mg]. Drug-related adverse events were reported in 89% of patients, the most common was neutropenia (47%). Seven patients had complete responses (CR), 7 had partial responses (PRs); 9 of 20 (45%) patients at the MRV RP2D responded (4 CRs, 5 PRs), all with non-germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL)., Conclusions: The combination of alisertib 50 mg b.i.d. plus rituximab or alisertib 40 mg b.i.d. plus rituximab and vincristine was well tolerated and demonstrated activity in non-GCB DLBCL., (©2018 American Association for Cancer Research.)

Background: This study was designed to determine the safety, tolerability, and pharmacokinetics (PK) of alisertib (MLN8237) in combination with docetaxel and to identify a recommended dose for the combination., Methods: Adults with metastatic cancer were treated on 21-day cycles with alisertib (10, 20, 30, or 40 mg) twice daily on days 1 to 7 or days 1 to 5 and with docetaxel (75 or 60 mg/m(2) ) on day 1. The primary objectives were to assess the safety and tolerability of the combination and to determine the recommended phase 2 dose (RP2D) for future studies. Secondary objectives included an efficacy assessment and PK analyses of docetaxel and alisertib., Results: Forty-one patients participated. Eight dose levels were explored with various doses of alisertib and docetaxel. The dose-limiting toxicities were neutropenic fever, neutropenia without fever, stomatitis, and urinary tract infection. The RP2D of this combination was 20 mg of alisertib twice daily on days 1 to 7 and intravenous docetaxel at 75 mg/m(2) on day 1 in 21-day cycles. Eight of the 28 patients (29%) who were efficacy-evaluable had objective responses. These included 1 complete response in a patient with bladder cancer, 6 partial responses in patients with castration-resistant prostate cancer, and 1 partial response in a patient with angiosarcoma. Concomitant administration of alisertib did not produce any clinically meaningful change in docetaxel PK., Conclusions: Alisertib at 20 mg twice daily on days 1 to 7 with intravenous docetaxel at 75 mg/m(2) on day 1 in a 21-day cycle was well tolerated, and the combination demonstrated antitumor activity. Cancer 2016;122:2524-33. © 2016 American Cancer Society., (© 2016 American Cancer Society.)

Purpose: Aurora A kinase (AAK) is overexpressed in aggressive lymphomas and can correlate with more histologically aggressive forms of disease. We therefore designed a phase II study of alisertib, a selective AAK inhibitor, in patients with relapsed and refractory aggressive non-Hodgkin lymphomas., Patients and Methods: Patients age ≥ 18 years were eligible if they had relapsed or refractory diffuse large B-cell lymphoma (DLBCL), mantle-cell lymphoma (MCL), transformed follicular lymphoma, Burkitt's lymphoma, or noncutaneous T-cell lymphoma. Alisertib was administered orally at 50 mg twice daily for 7 days in 21-day cycles., Results: We enrolled 48 patients. Histologies included DLBCL (n = 21), MCL (n = 13), peripheral T-cell lymphoma (n = 8), transformed follicular lymphoma (n = 5), and Burkitt's (n = 1). Most common grade 3 to 4 adverse events were neutropenia (63%), leukopenia (54%), anemia (35%), thrombocytopenia (33%), stomatitis (15%), febrile neutropenia (13%), and fatigue (6%). Four deaths during the study were attributed to progressive non-Hodgkin lymphoma (n = 2), treatment-related sepsis (n = 1), and unknown cause (n = 1). The overall response rate was 27%, including responses in three of 21 patients with DLBCL, three of 13 with MCL, one of one with Burkitt's lymphoma, two of five with transformed follicular lymphoma, and four of eight with noncutaneous T-cell lymphoma. The alisertib steady-state trough concentration (n = 25) revealed the expected pharmacokinetic variability, with a trend for higher incidence of adverse event-related dose reductions at higher trough concentrations. Analysis for AAK gene amplification and total AAK protein revealed no differences between histologies or correlation with clinical response., Conclusion: The novel AAK inhibitor alisertib seems clinically active in both B- and T-cell aggressive lymphomas. On the basis of these results, confirmatory single-agent and combination studies have been initiated.

RON is a receptor tyrosine kinase of the MET family that is involved in cell proliferation, cell survival, and cell motility in both normal and disease states. Macrophage-stimulating protein (MSP) is the RON ligand whose binding to RON causes receptor activation. RON is a trans-membrane heterodimer comprised of one alpha- and one beta-chain originating from a single-chain precursor and held together by several disulfide bonds. The intracellular part of RON contains the kinase domain and regulatory elements. The extracellular region is characterized by the presence of a sema domain (a stretch of approximately 500 amino acids with several highly conserved cysteine residues), a PSI (plexin, semaphorins, integrins) domain, and four immunoglobulin-like folds. Here we show that a soluble, secreted molecule representing the sema domain of RON (referred to as ron-sema) has a dominant negative effect on the ligand-induced receptor activation and is capable of inhibiting RON-dependent signaling pathways and cellular responses. Results suggest that the sema domain of RON participates in ligand binding by the full-length receptor. The ability of ron-sema to suppress growth of MSP-responsive cells in culture, including cancer cells, points to a potential therapeutic use of this molecule, and forced expression of it could potentially be used as a gene therapy tool for treating MSP-dependent types of cancer.

beta-Catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expression. Accumulation of cellular beta-catenin occurs in many types of human cancers. Four mechanisms are known to cause increases in beta-catenin: mutations of beta-catenin, adenomatous polyposis coli, or axin genes and activation of Wnt signaling. We report a new cause of beta-catenin accumulation involving oncogenic mutants of RON and MET receptor tyrosine kinases (RTKs). Cells transfected with oncogenic RON or MET were characterized by beta-catenin tyrosine phosphorylation and accumulation; constitutive activation of a Tcf transcriptional factor; and increased levels of beta-catenin/Tcf target oncogene proteins c-myc and cyclin D1. Interference with the beta-catenin pathway reduced the transforming potential of mutated RON and MET. Activation of beta-catenin by oncogenic RON and MET constitutes a new pathway, which might lead to cell transformation by these and other mutant growth factor RTKs.

Macrophage stimulating protein (MSP) is secreted as 78-kDa single chain pro-MSP, which is converted to biologically active, disulfide-linked alphabeta chain MSP by cleavage at Arg(483)-Val(484). Murine resident peritoneal macrophages have two cell surface proteolytic activities that cleave pro-MSP. One is a pro-MSP convertase, which cleaves pro-MSP to active MSP; the other degrades pro-MSP. The degrading protease is inhibited by soybean trypsin inhibitor or by low concentrations of blood plasma, which allows the convertase to cleave pro-MSP to MSP. Using pro-MSP cleavage as the assay, we purified the inhibitor from human plasma. The bulk of the plasma protein was removed by salting out and by isoelectric precipitation of albumin. Highly purified inhibitor was then obtained in three steps: dye-ligand binding and elution, ion exchange chromatography, and high performance liquid chromatography gel filtration. After SDS-polyacrylamide gel electrophoresis and transfer to a polyvinylidene membrane, N-terminal sequencing of the product identified it as alpha(1)-antichymotrypsin. The mean concentration of alpha(1)-antichymotrypsin in human plasma is 7 micrometer. At this concentration, alpha(1)-antichymotrypsin inhibits both macrophage enzymes. A concentration of 0.4 micrometer, which is in the expected concentration range in extracellular fluid, preferentially inhibits the degrading enzyme, which allows for cleavage to active MSP by the pro-MSP convertase.

MSP is a serum protein belonging to the plasminogen-related kringle domain protein family. In addition to macrophages, epithelial cells are also MSP targets. MSP is a multifunctional factor regulating cell adhesion and motility, growth and survival. MSP mediates its biological activities by activating a transmembrane receptor tyrosine kinase called RON in humans or SKT in mice. MSP can protect epithelial cells from apoptosis by activating two independent signals in the PI3-K/AKT or the MAPK pathway. The MAPK pathway mediates the MSP antiapoptotic effect only if additional signaling pathways are activated through adhesion. This indicates that MSP receptors and integrins, the receptors mediating cell-matrix-dependent adhesion, can collaborate in promotion of cell survival. This adhesion-dependent pathway, which is essential for the MAPK-mediated anti-apoptotic effect, remains to be identified. A hypothesis that Stat3 might represent a key component of the adhesion-induced anti-apoptotic pathway is presented in this review.

RON is a transmembrane receptor tyrosine kinase that mediates biological activities of Macrophage Stimulating Protein (MSP). MSP is a multifunctional factor regulating cell adhesion, motility, growth and survival. MSP binding to RON causes receptor tyrosine phosphorylation leading to up-regulation of RON catalytic activity and subsequent activation of downstream signaling molecules. Recent studies show that RON is spatially and functionally associated with other transmembrane molecules including adhesion receptors integrins and cadherins, and cytokine and growth factor receptors IL-3 betac, EPOR and MET. For example, MSP-induced cell shape change is mediated via RON-activated IL-3 betac receptor. Activation of integrins causes MSP-independent RON phosphorylation, and the integrin/RON collaboration regulates cell survival. Thus, RON can be activated without MSP by ligand stimulation of RON-associated receptors, and MSP-activated RON can cause ligand-independent activation of RON-associated receptors. As a result of the receptor cross-activation RON-specific pathways become a part of a signal transduction network of other receptors, and conversely signaling pathways activated by other receptors can be used by RON. This receptor collaboration extends the spectrum of cellular responses generated by MSP and by putative ligands of RON-associated receptors. However signaling pathways involved in the receptor cross-talk and underlying activation mechanisms remain to be investigated. The purpose of this review is to summarize data and to discuss a role of cross-talk between RON and other transmembrane receptors.

The human RON gene (MST1R) maps to 3p21.3, a region frequently altered in lung cancer and other malignancies. It encodes a receptor tyrosine kinase (RTK) closely related to MET, whose mutations are associated with neoplasia. We investigated whether RON might be involved in the development or progression of lung cancer. We first determined the exon-intron structure of the gene by direct sequencing of RON cosmid DNA and PCR products containing intronic sequences, and then developed primers suitable for mutation analysis by the single-strand conformation polymorphism (SSCP) method. Twenty coding exons were characterized, all but the first one small (average size: 170 bp), a feature shared with other RTK genes. We performed SSCP analysis of RON in small and non-small cell lung cancer samples, upon detection of its expression in a sample of lung cancer cell lines. A mutation (T915C: L296P) was found in an adenocarcinoma specimen. Several single nucleotide polymorphisms were also found. The panel of intron-anchored primers developed in this work will be useful for mutation analysis of the RON gene in different types of human tumors., (Copyright 2000 Wiley-Liss, Inc.)

Cooperation between integrins and growth factor receptors plays an important role in the regulation of cell growth, differentiation, and survival. The function of growth factor receptor tyrosine kinases (RTKs) can be regulated by cell adhesion to extracellular matrix (ECM) even in the absence of ligand. We investigated the pathway involved in integrin-mediated RTK activation, using RON, the receptor for macrophage-stimulating protein. Adhesion of RON-expressing epithelial cells to ECM caused phosphorylation of RON, which depended on the kinase activity of both RON itself and c-Src. This conclusion is based on these observations: 1) ECM-induced RON phosphorylation was inhibited in cells expressing kinase-inactive c-Src; 2) active c-Src could phosphorylate immunoprecipitated RON from ECM-stimulated cells but not from unstimulated cells; and 3) ECM did not cause RON phosphorylation in cells expressing kinase-dead RON, nor could active c-Src phosphorylate RON immunoprecipitated from these cells. The data fit a pathway in which ECM-induced integrin aggregation causes both c-Src activation and RON oligomerization followed by RON kinase-dependent autophosphorylation; this results in RON becoming a target for activated c-Src, which phosphorylates additional tyrosines on RON. Integrin-induced epidermal growth factor receptor (EGFR) phosphorylation also depended on both EGFR and c-Src kinase activities. This sequence appears to be a general pathway for integrin-dependent growth factor RTK activation.

In addition to its effects on macrophage function, macrophage-stimulating protein (MSP) is a growth and motility factor for epithelial cells. The growth and survival of epithelial cells generally require two signals, one generated by interaction with extracellular matrix via integrins, the other initiated by a growth factor. Therefore we investigated the effect of MSP on epithelial cell survival. Survival of epithelial cells cultured overnight in serum-free medium was promoted by adhesion, which activated both the phosphatidylinositol 3'-kinase (PI3-K)/AKT and mitogen-activated protein kinase (MAPK) pathways, operating independently of one another. The number of apoptotic cells resulting from inhibition of either pathway alone was approximately doubled by simultaneous inhibition of both pathways. This shows that each pathway made a partial contribution to the prevention of apoptosis. In the presence of an inhibitor of either pathway, MSP increased the activity of the other pathway so that the single uninhibited pathway alone was sufficient to prevent apoptosis. In contrast to the results with adherent cells, although MSP also prevented apoptosis of cells in suspension (anoikis), its effect was mediated only by the PI3-K/AKT pathway. Despite activation of MAPK by MSP, anoikis was not prevented in suspended cells with a blocked PI3-K/AKT pathway. Thus, activation of MAPK alone is not sufficient to mediate MSP antiapoptotic effects. Cell adhesion generates an additional signal, which is essential for MSP to use MAPK in an antiapoptotic pathway. This may involve translocation of MSP-activated MAPK from the cytoplasm into the nucleus, which occurs only in adherent cells. Our results suggest that there is cross talk between cell matrix adhesion and growth factors in the regulation of cell survival via the MAPK pathway. Growth factors induce MAPK activation, and adhesion mediates MAPK translocation from the cytoplasm into the nucleus.

Macrophage-stimulating protein (MSP) and hepatocyte growth factor/scatter factor (HGF/SF) are plasminogen-related growth and motility factors that interact with cell-surface protein tyrosine kinase receptors. Each one is a heterodimeric protein comprising a disulfide-linked alpha chain and a serine protease-like beta chain. Despite structural similarities between MSP and HGF, the primary receptor binding site is located on the alpha chain of HGF/SF but on the beta chain of MSP. To obtain insight into the structural basis for MSP beta chain binding, beta chain structure was modeled from coordinates of an existing model of the HGF beta chain. The model revealed that the region corresponding to the S1 specificity pocket in trypsin is filled by the Asn(682)/Glu(648) interacting pair, leaving a shallow cavity for possible beta chain interaction with the receptor. Mutants in this region were created, and their binding characteristics were determined. A double mutation of Asn(682)/Glu(648) caused diminished binding of the beta chain to the MSP receptor, and a single mutation of neighboring Arg(683) completely abolished binding. Thus, this region of the molecule is critical for binding. We also found that at equimolar concentrations of free alpha and beta chains, alpha chain binding to receptor was detectable, at levels considerably lower than beta chain binding. The EC(50) values determined by quantitative enzyme-linked immunosorbent assay are 0.25 and 16.9 nM for beta and alpha chain, respectively. The data suggest that MSP has two independent binding sites with high and low affinities located in beta and alpha chain, respectively, and that the two sites together mediate receptor dimerization and subsequent activation.

Macrophage stimulating protein (MSP) is a growth and motility factor that mediates its activity via the RON/STK receptor tyrosine kinase. MSP promotes integrin-dependent epithelial cell migration, which suggests that MSP may regulate integrin receptor functions. Integrins are cell surface receptors for extracellular matrix. Epithelial cell adhesion and motility are mediated by integrins. We studied the enhancement by MSP of cell adhesion and the molecular mechanisms mediating this effect. MSP decreased the time required for adhesion of 293 and RE7 epithelial cells to substrates coated with collagen or fibronectin. Prevention of adhesion by an RGD-containing peptide showed that the cell-substrate interaction was mediated by integrins. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), blocked MSP-dependent adhesion, which shows that PI3-K is in the MSP-induced adhesion pathway. MSP also affected focal adhesion kinase (FAK) which is important for some types of cell adhesion and motility. Although MSP caused PI3-K-independent tyrosine phosphorylation and activation of FAK, experiments with dominant-negative FAK constructs showed that FAK does not mediate the effects of MSP on cell adhesion or motility. Thus PI3-K, but not FAK, mediates MSP-induced integrin-dependent adhesion of epithelial cells. Also, we found ligand-independent association between RON and beta1 integrin, which is additional evidence for a relationship between these two receptor systems.

Human serum macrophage-stimulating protein (MSP) induces motile activity of murine resident peritoneal macrophages and is a growth and motility factor for epithelial cells. It belongs to the plasminogen-related family of kringle proteins, and is secreted as a single-chain, 78-kDa, biologically inactive pro-MSP. Proteolytic cleavage of pro-MSP at a single site yields active MSP, a disulfide-linked alphabeta-chain heterodimer. However cleavage of recombinant pro-MSP yielded not only the disulfide-linked heterodimer, but also free alpha- and beta-chains, indicating that some of the recombinant molecules lacked an alphabeta-chain disulfide. We purified the free chains for characterization. The beta-chain of MSP has three extra cysteines, Cys527, Cys562, and Cys672, which are not found in the plasminogen beta-chain. Disulfide bond analysis showed a Cys527-Cys562, but also a Cys588-Cys672. Coopting Cys588 by Cys672 prevented the expected formation of a disulfide between alpha-chain Cys468 and beta-chain Cys588. Concomitant studies determined structures of oligosaccharides at the three Asn-linked glycosylation sites of MSP. The oligosaccharides at the three Asn loci are heterogeneous; 11 different sugars were identified, all being sialylated fucosyl biantennary structures. We also located the pro-MSP signal peptide cleavage site at Gly18-Gln19 and the scissile bond for formation of mature MSP at Arg483-Val484., (Copyright 1999 Academic Press.)

Macrophage stimulating protein (MSP) belongs to the plasminogen-related kringle domain family. In addition to stimulation of macrophages, MSP acts on other cell types including epithelial and hematopoietic cells. The MSP receptor is a transmembrane tyrosine kinase called RON in humans and STK in mice. MSP/receptor interaction induces activation of signal transduction pathways that mediate MSP biological activities. Cytoplasmic kinases are intracellular messengers occupying an important role in signal transduction. We have identified kinases that participate in RON signaling. In addition to previously identified involvement of phosphatidylinositol 3-kinase (PI3-K), JNK, and MAPK, we found that FAK, c-Src, and AKT are rapidly and transiently activated by MSP. FAK, MAPK, and c-Src are involved in MSP-induced cell proliferation. MAPK and c-Src are components of one signal transduction cascade, and MAPK is downstream of c-Src. FAK also regulates MSP-induced cell growth, but via a path different from c-Src/MAPK. AKT kinase is a component of a separate branch of the RON/PI3-K pathway that mediates the MSP anti-apoptotic effect on epithelial cells. PI3-K regulates MSP-induced adhesion and motility but via downstream components different from AKT. Thus, occupancy of the RON receptor by MSP activates distinct signal transduction pathways that mediate several cellular responses.

Macrophage stimulating protein (MSP) exists in blood as inactive pro-MSP. Cleavage yields active MSP, the ligand for a membrane receptor (RON) that is expressed on keratinocytes as well as macrophages. Because both cells have roles in tissue injury, we looked for active MSP and expressed RON in wounds. Concentration of pro-MSP + MSP in wound exudates was in the range for optimal activity. Western blot showed that MSP comprised about half the total, in contrast to less than 10% of the total in blood plasma. The presence of MSP was attributed to an exudate pro-MSP convertase that had an inhibitor profile consistent with a trypsin-like serine protease. Exudate evoked morphologic changes in macrophages in vitro like that of MSP. Removal of this activity by an anti-MSP column shows that exudate stimulation of macrophages is due to MSP. RON was infrequently detected in normal skin. RON protein was markedly upregulated in burn wound epidermis and accessory structures, in proliferating cells or differentiated cells, or both. RON was also detected on macrophages and capillaries. Tissue injury leads to cleavage of pro-MSP to MSP, which has potential to act on keratinocytes, macrophages, and capillaries, all components of the wound healing response.

Hepatocyte growth factor/scatter factor (HGF/SF) and macrophage stimulating protein (MSP) are plasminogen-related kringle proteins that lost serine protease domain enzymatic activity and became ligands for cell surface tyrosine kinase receptors. They are activated by cleavage to disulfide-linked alphabeta chains. Surprisingly, despite structural similarities, the high affinity receptor binding regions of the two proteins are different: alpha chain for HGF, and beta chain for MSP. We propose that after cleavage exposes a beta chain binding site (high affinity for MSP, low affinity for HGF), monomeric ligand induces receptor dimerization and activation via alpha and beta chain binding sites of different affinity.

Macrophage stimulating protein (MSP) is a 78-kDa disulfide-linked heterodimer belonging to the plasminogen-related kringle protein family. MSP activates the RON receptor protein-tyrosine kinase, which results in cell migration, shape change, or proliferation. A structure-activity study of MSP was performed using pro-MSP, MSP, MSP alpha and beta chains, and a complex including the first two kringles and IgG Fc (MSP-NK2). Radioiodinated MSP and MSP beta chain both bound specifically to RON. The Kd of 1.4 nM for MSP beta chain is higher than the reported Kd range of 0.6-0.8 nM for MSP. Pro-MSP, MSP alpha chain, and MSP-NK2 did not bind. Only MSP stimulated RON autophosphorylation. Although the beta chain bound to RON and partially inhibited MSP-induced RON phosphorylation in kidney 293 cells, it did not induce RON phosphorylation. Pro-MSP, MSP alpha chain, or MSP-NK2 failed to activate RON, consistent with their inability to bind to the RON receptor. Functional studies showed that only MSP induced cell migration, and shape change in resident macrophages, and growth of murine keratinocytes. Our data indicate that the primary receptor binding domain is located in a region of the MSP beta chain, in contrast to structurally similar hepatocyte growth factor, in which the receptor binding site is in the alpha chain. However, full activation of RON requires binding of the complete MSP disulfide-linked alphabeta chain heterodimer.

Macrophage-stimulating protein (MSP; also known as HGF-like protein [HGFl]) is a 78 kDa plasma protein that is secreted by the liver into the circulation as single-chain, biologically inactive pro-MSP. The presence of conserved triple disulfide loops (kringles) places pro-MSP in a family of coagulation system serine protease zymogens that are activated by proteolytic cleavage. Although pro-MSP has lost enzymic activity, it has retained the activation mechanism, in that proteolytic cleavage at a single site yields biologically active disulfide-linked alpha beta-chain heterodimeric MSP. The MSP receptor is a transmembrane protein tyrosine kinase. MSP causes phosphorylation of the receptor cytoplasmic domain, association of phosphatidylinositol (PI)-3 kinase with the receptor, and phosphorylation of receptor-bound PI-3 kinase. Inhibition of PI-3 kinase by wortmannin prevents MSP action on cells. MSP stimulates motility of murine resident peritoneal macrophages. However, it does not act on exudate macrophages or blood monocytes, since these earlier maturational stages of the lineage do not express the receptor. MSP also stimulates keratinocyte cell lines, causing either chemotactic responses or increased cell numbers in culture. We suggest that pro-MSP diffuses into local tissue sites, where proteolytic cleavage to MSP results in stimulation of keratinocytes and macrophages. It possibly plays a role in tissue injury or wound healing.

Macrophage stimulating protein (MSP) is a ligand for the RON receptor protein tyrosine kinase. Activation of RON in murine resident macrophages results in cell shape change and migration. We studied cell movement induced by MSP in different types of human epithelial cells and the possible role of phosphatidylinositol-3 (PI-3) kinase in RON-mediated signal transduction. We observed specific and saturable binding of 125I-MSP to RON on several epithelial cell lines. In addition to activation and phosphorylation of RON, MSP also induced tyrosine phosphorylation of the PI-3 kinase p85 subunit in a time-dependent manner, with a peak at 15 min. Moreover, phosphorylated RON formed a complex with PI-3 kinase in both HK-NOC keratinocyte and RON cDNA-transfected MDCK cells. An in vitro protein interaction assay confirmed that PI-3 kinase from a lysate of MSP-activated cells bound to pure RON protein. MSP, at a concentration range of 1 to 5 nM, induced migration of three epithelial cell lines. This effect was inhibited by wortmannin, a specific inhibitor for PI-3 kinase, with an IC50 of 10 nM. MSP-induced shape change in murine resident peritoneal macrophages was also abolished by wortmannin. These data suggest that activation of PI-3 kinase is required for MSP-induced epithelial cell migration. The stimulation by MSP of epithelial cell movement may have implications for tissue repair, wound healing, and tumor metastasis.

We injected 125I-pro-macrophage-stimulating protein (pro-MSP) intravenously into normal mice to determine its clearance from the circulation and to test for conversion of pro-MSP to the biologically active heterodimer in the absence of inflammation or tissue injury. Pro-MSP was cleared from the circulation with a half-life of approximately 100 min. This rapid clearance was not peculiar to 125I-pro-MSP, since clearance rates of unlabeled pro-MSP and of 125I-bovine serum albumin were comparable. The liver was the major locus of radioactivity 10-20 min after the intravenous injection of 125I-pro-MSP. By 90 min, over 60% of total recovered radioactivity was in the small intestine. Reflecting gastrointestinal transit, counts decreased in the small intestine and appeared in the colon by 180 min. Essentially all counts in urine and feces obtained at later times were soluble in trichloracetic acid. These findings reflected rapid hepatic proteolysis of pro-MSP to fragments undetectable by antibody to pro-MSP; within 20 min after intravenous administration, immunoprecipitable counts were only 22% of the total liver extract radioactivity. Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioautography data for immunoprecipitated plasma and liver extract revealed no evidence for hepatic conversion of pro-MSP to MSP. Thus, the hepatic catabolic pathway of pro-MSP is degradative and does not yield mature MSP. The results support our view that MSP is not released into the circulation but is generated at specific extravascular loci by pro-MSP convertases.

Free cytokine and chemokine concentrations in normal human serum are generally below the detection limit. For IL-8, this limit is about 6 pm. In one study, with an ELISA of higher sensitivity, mean serum IL-8 concentration was 2 ± 0.2 pm. Measurement of autoantibody and immune complex in normal human serum suggests a distinction between cytokines and chemokines. Free serum autoantibody to IL-1alpha, TNF-alpha, IFN-gamma, and IL-6 have been found, whereas serum cytokine immune complexes have generally not been described. In contrast, IL-8 and MCP-1 immune complexes have been detected in all normal sera (mean concentrations for 48 sera of 220 and 330 pm, respectively), whereas free autoantibody is not found in the majority of normal sera. Analysis of MCP-1 immune complexes indicates that the antibody is of high affinity, which is consistent with our inability to detect free antigen in the serum. It will be of interest to look for other chemokine serum immune complexes, to determine if the data for IL-8 and MCP-1 apply to chemoattractant cytokines generally. A central question is whether autoantibody plays a regulatory role as a sink for circulating cytokine or chemokine. This can perhaps be answered by testing for cytokine activity of immune complexes.

Macrophage stimulating protein (MSP) is a chemotactic factor for murine peritoneal macrophages. The receptor for human MSP was recently identified as the ron gene product, a transmembrane protein tyrosine kinase cloned from a human keratinocyte cDNA library. Here we report that MSP induced proliferation of murine primary keratinocytes and established keratinocyte cell lines in a concentration-dependent manner. The growth efficacy of MSP was comparable to that of epidermal growth factor and keratinocyte growth factor. In three of four cell lines tested in a chemotaxis chamber, MSP also stimulated migration of keratinocytes on a collagen type IV substratum. The action of MSP was mediated by specific binding of MSP to the STK gene product, a murine homologue of the RON MSP receptor. Binding of MSP to keratinocyte STK induced phosphorylation of the 150 kDa STK beta chain. Herbimycin A, a protein tyrosine kinase inhibitor, blocked MSP-mediated phosphorylation of the STK receptor as well as proliferation of keratinocytes, suggesting the importance of tyrosine kinase activity for transduction of the message delivered by MSP. Previously, the only known target cell for MSP was the resident peritoneal macrophage. These studies establish the keratinocyte as a new target cell for MSP. The action of MSP on keratinocytes may have implications for tissue repair, wound healing, and tumor growth.

Macrophage-stimulating protein (MSP), originally identified as an inducer of murine resident macrophage responsiveness to chemoattractants, is a ligand for human RON/murine STK receptor protein tyrosine kinases. Since STK was cloned from populations enriched for hematopoietic stem cells, we initiated studies on the effects of MSP on colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) myeloid progenitor cells. MSP alone had no colony stimulating activity. However, MSP caused about a 50% suppression of CFU-GM colony formation induced by synergistic combinations of SLF or Flt-L plus GM-CSF, G-CSF, or IL-3 and of BFU-E and CFU-GEMM colonies induced by SLF or Flt3-L plus Epo or Epo and IL-3. In contrast, MSP had no effect on progenitors stimulated by one growth factor. MSP also suppressed colony formation by stimulated cord blood progenitors, but only after preinduction to a rapidly cycling state. It was previously reported that several members of the chemokine family synergistically suppress myeloid progenitor proliferation. Likewise, synergistic suppression was observed when MSP was paired with VEGF, MIP-1 alpha, IL-8, PF4, MCP-1, IP-10, or ENA-78, or when VEGF was paired with the chemokines; and the required MSP concentration was more than 100-fold less than for MSP alone. Additionally, MSP or VEGF inhibited proliferation of the human myeloid growth factor-dependent cell line, M07e, but a sustained effect required multiple additions over time. At the least, some of the MSP suppressive effects on myeloid progenitors, as assessed on single isolated CD34 marrow cells, appeared to be directly on the progenitors; sustained additions of MSP were required to see this effect. The suppressive action of MSP and its synergism with proteins of the chemokine family may be of relevance to regulation of blood cell production.

Macrophage stimulating protein (MSP), which is secreted as biologically inactive pro-MSP, is activated to MSP by cleavage at a single peptide bond. Our objectives were to determine the form of MSP in circulating blood and to study proteolytic activation of pro-MSP by its target cell. Western blot of immunoaffinity-purified serum MSP showed that all the protein was pro-MSP, without detectable MSP. The circulating form of the protein is therefore pro-MSP, and conversion to MSP does not occur when blood is shed. Incubation of radiolabeled pro-MSP with murine peritoneal macrophages caused proteolytic cleavage to predominantly inactive fragments. Among several protease inhibitors, soybean trypsin inhibitor was one of two that inhibited nonspecific cleavage and revealed a macrophage proteolysis of pro-MSP, and certain concentrations enhanced cleavage to mature MSP. Macrophage membranes had nonspecific and specific pro-MSP proteolytic activity, which was not present in macrophage culture fluids. The results suggest that control of MSP activity can occur at the level of the target cell by proteolytic cleavage of pro-MSP to mature MSP or to inactive fragments.

Macrophage-stimulating protein (MSP) is a chemotactic factor that activates the receptor tyrosine kinase RON. The involvement of Ras in MSP-induced signal transduction was investigated. Here we demonstrate that, in RON-transfected MDCK cells, an active GTP-bound form of Ras was rapidly accumulated by MSP treatment and the Ras-guanine nucleotide exchange activity in SOS immunoprecipitates was concomitantly increased. GAP activity was not changed under the same conditions used. Furthermore, the SH2 domain of adaptor protein GRB2, but not Shc, associated with the activated RON-beta chain, and GRB2-SOS complexes translocated from the cytosol to the membrane upon MSP treatment. These results strongly suggest that MSP activates Ras through RON, and that MSP-induced activation of Ras might be controlled by both the enhancement of catalytic exchange activity of SOS and its translocation to the membrane where its target Ras is localized.

Macrophage-stimulating protein (MSP) was originally identified as an inducer of murine resident peritoneal macrophage responsiveness to chemoattractants. We recently showed that the product of RON, a protein tyrosine kinase cloned from a human keratinocyte library, is the receptor for MSP. Similarity of murine stk to RON led us to determine if the stk gene product is the murine receptor for MSP. Radiolabeled MSP could bind to NIH 3T3 cells transfected with murine stk cDNA (3T3/stk). Binding was saturable and was inhibited by unlabeled MSP but not by structurally related proteins, including hepatocyte growth factor and plasminogen. Specific binding to STK was demonstrated by cross-linking of 125I-labeled MSP to membrane proteins of 3T3/stk cells, which resulted in a protein complex with a molecular mass of 220 kDa. This radiolabeled complex comprised 125I-MSP and STK, since it could be immunoprecipitated by antibodies to the STK beta chain. Binding of MSP to stk cDNA-transfected cells induced tyrosine phosphorylation of the 150-kDa STK beta chain within 1 min and caused increased motile activity. These results establish the murine stk gene product as a specific transmembrane protein tyrosine kinase receptor for MSP. Inasmuch as the stk cDNA was cloned from a hematopoietic stem cell, our data suggest that in addition to macrophages and keratinocytes, a cell in the hematopoietic lineage may also be a target for MSP.

Sweating in humans is induced by physical or emotional stress, which raises the possibility that sweating may relate to host defense. We therefore asked whether human eccrine sweat attracts leukocytes and found that it is chemotactic for human neutrophils. This activity was due to several chemoattractants, one of which was interleukin-8 (IL-8). Using immunohistochemistry and in situ hybridization IL-8 and its mRNA have been detected in sweat gland epithelium, indicating that IL-8 is produced in situ. This establishes a pattern of physiological IL-8 secretion by exocrine glands and suggests that, in addition to its role as a major inflammatory mediator, IL-8 also has physiological homeostatic functions.

Macrophage-stimulating protein (MSP) is a member of the hepatocyte growth factor-scatter factor (HGF-SF) family. Labeled MSP bound to Madin-Darby canine kidney (MDCK) cells transfected with complementary DNA encoding Ron, a cell membrane protein tyrosine kinase. Cross-linking of 125I-labeled MSP to transfected cells (MDCK-RE7 cells) and immunoprecipitation by antibodies to Ron revealed a 220-kilodalton complex, a size consistent with that of MSP (80 kilodaltons) cross-linked to the beta chain of Ron (150 kilodaltons). The binding of 125I-labeled MSP to MDCK-RE7 cells was inhibited by unlabeled MSP, but not by HGF-SF. MSP caused phosphorylation of the beta chain of Ron and induced migration of MDCK-RE7 cells. These results establish the ron gene product as a specific cell-surface receptor for MSP.

Human serum macrophage-stimulating protein (MSP) is a disulfide-linked heterodimer that induces motile and phagocytic activity of mouse resident peritoneal macrophages. In this work, we found that MSP blocked the increase in macrophage nitric oxide synthase mRNA, as well as the associated increase in nitric oxide production, that occurred in response to several stimuli. These included bacterial products and mammalian cytokines: endotoxin, and interferon-gamma plus endotoxin, interleukin-2, or tumor necrosis factor-alpha. The inhibition by MSP of induction of nitric oxide synthase mRNA and nitric oxide secretion was concentration-dependent. The concentration of MSP that caused maximal inhibition of nitric oxide production was comparable with the optimum for stimulation of macrophage motile and phagocytic activity. Time course studies showed that nitrite was first detected in culture fluid about 8 h after endotoxin stimulation, and it accumulated at a linear rate during the ensuing 16 h. Inhibition by MSP occurred during the 8-h lipopolysaccharide (LPS) induction period; inhibition was maximal when MSP and LPS were added together and decreased progressively to no inhibition as the interval between LPS and MSP addition increased to 11 h.