1. Introduction
Hepatocyte growth factor (HGF) and its receptor MET play roles in embryonic development and the repair of tissues following injury [
1,
2,
3]. HGF is a fibroblast-derived morphogen that induces epithelial branching duct formation [
4,
5]. Scatter factor, a fibroblast-derived cell motility factor for epithelial cells, is identical to HGF [
6]. In previous studies, the secretion of HGF was not noted in epithelial cells, but was in fibroblasts, vascular smooth muscle cells, and hematopoietic cells in culture [
7,
8]. Subsequent studies revealed the roles of HGF in the growth and morphogenesis of different types of epithelial cells and tissues as a mediator of epithelial–mesenchymal interactions [
9,
10,
11,
12,
13].
Mice lacking HGF were found to exhibit embryonic lethality due to impaired development of the placenta and liver [
14,
15]. In addition, MET-deficient mice had no skeletal muscle in the limbs and diaphragm because of the impaired migration of myogenic precursor cells to the limb buds and diaphragm [
16]. The characterization of conditional MET-deficient mice via analyses on a variety of cell types revealed that the HGF–MET pathway promotes tissue regeneration and protection, and suppresses the progression of chronic inflammation and fibrosis [
3,
17,
18,
19,
20].
HGF is a glycosylated protein composed of α- and β-chains linked by a disulfide bridge. It is biosynthesized and secreted as single-chain HGF (scHGF); the cleavage of scHGF at the Arg494–Val495 generates two-chain HGF (tcHGF) [
21,
22]. scHGF is a biologically inactive precursor incapable of activating the MET receptor, while tcHGF is the only active molecular species that can activate it. Previous studies indicated that HGF exists as scHGF in normal tissues and that its processing into tcHGF occurs in injured tissues [
23] and the tumor microenvironment [
21,
24]. Aberrant activation of the HGF–MET pathway, including ligand-dependent MET activation, is strongly associated with invasive growth, metastasis, and resistance to anticancer drugs [
2,
22].
These previous findings suggest that the localization of tcHGF is key to understanding how the HGF–MET pathway participates in tissue development, regeneration, and cancer progression. However, there are currently no molecular tools that specifically recognize tcHGF but not scHGF. Because the stomach is a typical organ composed of epithelial and mesenchymal cells, and stem cells play important roles in development and regeneration [
25], analyses of the localization of sc/tcHGF and its association with MET activation in the stomach may provide a better understanding of how the HGF–MET pathway participates in development, regeneration, and the stem cell behavior of endoderm-derived organs. In a recent study, we obtained several anti-HGF monoclonal antibodies that recognize different epitopes of human HGF [
26], one of which selectively recognizes human tcHGF. In this study, we thus investigated the localization of tcHGF and active MET in the developing and regenerating stomach, employing human tcHGF-specific monoclonal antibody and human HGF-knock-in mice.
3. Discussion
A variety of bioactive molecules are secreted or stored as inactive forms and activated in response to developmental programs, injury, and/or pathology. Proteolytic conversion of inactive scHGF to active tcHGF has a key role in the activation of HGF–MET signaling; however, because of the lack of a molecular tool able to selectively capture tcHGF, the spatial and temporal regulation of tcHGF generation in development and tissue repair has remained unknown. In this study, we observed a change in the localization of tcHGF during gastric development, homeostasis, and regeneration by using a human tcHGF-specific monoclonal antibody and hHGF-ki mice. tcHGF was localized in areas that differ both from the primary site of synthesis and the localization of scHGF. Conversely, tcHGF co-localized with activated MET.
Previous studies suggested that HGF is a mesenchymal-derived factor that plays a role in epithelial cell growth, movement, and branched tube formation. In the developing stomach, we found that scHGF was mainly localized in smooth muscle cells, whereas tcHGF was diffusely distributed in sub-epithelial cells and also localized in epithelial cells. Our finding that the localization of tcHGF but not scHGF was closely related to pMET localization agrees with previous studies. Our work also indicated that the conversion of scHGF into tcHGF occurred in sub-epithelial and epithelial cells and that tcHGF acted on MET-expressing epithelial cells. Because glycosaminoglycans and collagens serve as binding sites for HGF in the extracellular matrix [
30,
31], scHGF was moderately diffusible, and was converted to tcHGF in the pericellular microenvironment.
The proteolytic processing of scHGF to tcHGF is catalyzed by several serine proteases [
32]. Matriptase, a membrane-type serine protease, is broadly expressed in a variety of epithelial cells including human gastric epithelia [
33,
34]. The ablation of matriptase resulted in the impaired integrity and barrier function of epithelial tissues in the large intestine and epidermis [
35,
36]. HGF-activator is synthesized mainly in the liver and at a lower level in gastrointestinal tissue [
32,
37,
38]. HGF-activator present in the plasma is activated by thrombin in injured tissues. Mice deficient in HGF-activator showed no abnormal tissue homeostasis, but the injured mucosa was not sufficiently covered by regenerated epithelium following colitis-induced injury [
39]. A recent study reported that HGF-activator induced Go-to-G
Alert cell-cycle transition in stem cells in various tissues [
40]. Taking these findings together, matriptase and HGF-activator are potential serine proteases responsible for tcHGF generation in the stomach.
Lgr5
+ stem cells in gastrointestinal organs play important roles in homeostasis, regeneration, and cancer development [
41]. The Lgr5
+ stem cell-specific disruption of
MET indicated that the MET receptor is dispensable for the normal homeostasis of intestinal epithelial cells [
20]. However, MET deficiency in intestinal stem cells mildly attenuated intestinal regeneration after radiation-induced injury, presumably by impairing stem cell fitness [
20]. The involvement of the HGF–MET pathway in regeneration was also supported by intestinal crypt organoid cultures, where HGF–MET signaling induced the outgrowth of intestinal stem cells into mini-guts, with an equivalent potency to EGF [
20]. In addition, tumor microenvironment-derived HGF augmented Wnt/β-catenin signaling and clonogenicity in colon cancer stem cells characterized by high Wnt reporter activity [
42]. Our study revealed that pMET and tcHGF were localized in the base region of the gastric corpus gland, and MET activation occurred in Lgr5
+ gastric stem cells. Moreover, HGF induced organoid formation by gastric stem cells at a level comparable to that of EGF. Taken together, these findings show that although HGF is dispensable for the normal homeostasis of gastric stem cells, potentially because of redundant growth and survival signals, the HGF-MET pathway plays a supportive role in stem cell fitness, growth, and maintenance, and in stem cell-derived epithelial tissue expansion.
The HGF–MET pathway participates in homeostasis and regeneration in a variety of tissues. Studies of conditional MET-deficient mice have revealed that MET activation is important for the regeneration of organs such as the liver and skin [
17,
18,
19], although the extent of its contribution depends on the tissue type. Ligand-dependent MET activation is closely associated with the acquisition of resistance to anticancer drugs in the cancer microenvironment [
22]. Elucidation of the spatial and temporal patterns of tcHGF generation should promote our understanding of the precise mechanisms involved in tissue regeneration, stem cell activation, and cancer progression. To this end, molecular tools that selectively capture tcHGF should provide experimental opportunities to investigate these mechanisms.