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. 2017 Sep 7;12(9):e0184154.
doi: 10.1371/journal.pone.0184154. eCollection 2017.

SCF-KIT Signaling Induces endothelin-3 Synthesis and Secretion: Thereby Activates and Regulates endothelin-B-receptor for Generating Temporally- And Spatially-Precise Nitric Oxide to Modulate SCF- And or KIT-expressing Cell Functions

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SCF-KIT Signaling Induces endothelin-3 Synthesis and Secretion: Thereby Activates and Regulates endothelin-B-receptor for Generating Temporally- And Spatially-Precise Nitric Oxide to Modulate SCF- And or KIT-expressing Cell Functions

Lei L Chen et al. PLoS One. .
Free PMC article


We demonstrate that SCF-KIT signaling induces synthesis and secretion of endothelin-3 (ET3) in human umbilical vein endothelial cells and melanoma cells in vitro, gastrointestinal stromal tumors, human sun-exposed skin, and myenteric plexus of human colon post-fasting in vivo. This is the first report of a physiological mechanism of ET3 induction. Integrating our finding with supporting data from literature leads us to discover a previously unreported pathway of nitric oxide (NO) generation derived from physiological endothelial NO synthase (eNOS) or neuronal NOS (nNOS) activation (referred to as the KIT-ET3-NO pathway). It involves: (1) SCF-expressing cells communicate with neighboring KIT-expressing cells directly or indirectly (cleaved soluble SCF). (2) SCF-KIT signaling induces timely local ET3 synthesis and secretion. (3) ET3 binds to ETBR on both sides of intercellular space. (4) ET3-binding-initiated-ETBR activation increases cytosolic Ca2+, activates cell-specific eNOS or nNOS. (5) Temporally- and spatially-precise NO generation. NO diffuses into neighboring cells, thus acts in both SCF- and KIT-expressing cells. (6) NO modulates diverse cell-specific functions by NO/cGMP pathway, controlling transcriptional factors, or other mechanisms. We demonstrate the critical physiological role of the KIT-ET3-NO pathway in fulfilling high demand (exceeding basal level) of endothelium-dependent NO generation for coping with atherosclerosis, pregnancy, and aging. The KIT-ET3-NO pathway most likely also play critical roles in other cell functions that involve dual requirement of SCF-KIT signaling and NO. New strategies (e.g. enhancing the KIT-ET3-NO pathway) to harness the benefit of endogenous eNOS and nNOS activation and precise NO generation for correcting pathophysiology and restoring functions warrant investigation.

Conflict of interest statement

Competing Interests: The commercial affiliations (i.e. ARUP Laboratories and Vel-Lab Research) does not alter our adherence to PLoS ONE policies on sharing data and materials.


Fig 1
Fig 1. Enzyme-linked immunosorbent assay (ELISA) and in situ immunohistochemistry demonstrate induction of endothelin-3 by SCF-KIT signaling.
(A), HUVECs responded to SCF with significant synthesis of ET3 (left panel) and secretion of ET3 in media (right panel). (B), WM793 melanoma cells responded to SCF with significant synthesis (left panel) and secretion of ET3 in serum-free culture medium (right panel). (C), In situ IHC. Top panels, control WM793 cells without SCF stimulation, more than 95% cells exhibited completely negative staining for BigET3, ECE-1, or ET3. Lower panels, WM793 cells after stimulation with SCF (100 ng/ml) for 24 hours. ETBR expression remained unchanged (a and e). Robust induction of BigET3 (f), ECE-1 (g), ET3 (h), and clusters of sub-membranous ET3 before secretion (h, open arrows) are observed in more than 95% of cells comparing to the respective controls (b, c, d).
Fig 2
Fig 2. Autophosphorylation, internalization, and nuclear localization of activated KIT with tyrosine phosphorylation at 568/570 (pY568/pY570KIT).
(A), IHC of frozen sections of an aggressive GIST (a-c) and a normal human adult testis as external control (d-f) using pan-KIT antibody (a and d), pY568/pY570KIT antibody (b and e, red arrow indicates nuclear localization), and pY703KIT antibody (c and f) respectively. (B), In situ IHC to assess kinetics of SCF-induced nuclear translocation of pY568/pY570KIT using WM793 melanoma cells cultured in 4-well chamber tissue culture treated glass slides. Control (g) without SCF stimulation, after addition of SCF to culture media, the nuclear localization of pY568/pY570KIT increases progressively (h-j) in more than 90% of WM793 cells, reaches a plateau about 40–60 minutes (i and j), begins to decrease at 90 minutes (k), and is completely absent in nucleus with relocation back to the cytoplasm at 4 hours, some residual cytoplasmic staining remains visible (l).
Fig 3
Fig 3. KIT activation & up-regulation, concomitant parallel induction of ET3, KIT+Melan-A- progenitor cells, and melanocyte regeneration in proportion to sun-exposure.
(A), IHC of KIT and ET3 on serial sections of human skin specimen obtained from a lower extremity-amputation. Sole represents active suppression of melanogenesis (a and d), dorsum of big toe represents intermediate sun-exposure (b and e), and lateral lower leg represents heavy sun-exposure (c and f). (B), IHC of KIT, Melan-A, and ET3 on serial sections of human skin punch biopsy specimens obtained from sun-protected axilla (g, i, k) and chronic heavy sun-exposed forearm (h, j, l) from the same individual. Lymphocytes serve as internal negative control for KIT, ET3 and Melan-A; mast cells serve as internal positive control for KIT. Together, these images demonstrate that human skin exhibits sun-exposure-dependent up-regulation of KIT (a-c) and concomitant parallel sun-exposure-induced increasing induction of ET3 (d-f). Chronic sun-exposure induces intense dendritic pattern of KIT expression as well as a large increase in the number of KIT-expressing-cells in the basal layer (h) consisting of KIT+Melan-A+ mature melanocytes (j) and KIT+Melan-Amelanocyte progenitor cells as evidenced by the difference between (h) and (j).
Fig 4
Fig 4. Immunohistochemical studies on human colon myenteric plexus demonstrate that 48 hours fasting results in activation of SCF-KIT signaling and concomitant parallel induction of endothelin-3.
Human colon specimens post 48 hours fasting demonstrate intense KIT staining in ICCs within the myenteric plexus (a), and intense ET3 staining within the myenteric plexus (b). In sharp contrast, the surrounding longitudinal and circular smooth muscle cells (SM) show negative ET3 staining.

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The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The commercial affiliations provided support in the form of salaries for authors [JS, SRT, AJ, MAV], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.