The growth, survival, and metabolic activities of multicellular organisms at the cellular level are regulated by intracellular signaling, systemic homeostasis and the pericellular microenvironment. Pericellular proteolysis has a crucial role in processing bioactive molecules in the microenvironment and thereby has profound effects on cellular functions. Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and HAI-2 are type I transmembrane serine protease inhibitors expressed by most epithelial cells. They regulate the pericellular activities of circulating hepatocyte growth factor activator and cellular type II transmembrane serine proteases (TTSPs), proteases required for the activation of hepatocyte growth factor (HGF)/scatter factor (SF). Activated HGF/SF transduces pleiotropic signals through its receptor tyrosine kinase, MET (coded by the proto-oncogene MET), which are necessary for cellular migration, survival, growth and triggering stem cells for accelerated healing. HAI-1 and HAI-2 are also required for normal epithelial functions through regulation of TTSP-mediated activation of other proteases and protease-activated receptor 2, and also through suppressing excess degradation of epithelial junctional proteins. This review summarizes current knowledge regarding the mechanism of pericellular HGF/SF activation and highlights emerging roles of HAIs in epithelial development and integrity, as well as tumorigenesis and progression of transformed epithelial cells.

Hepatocyte growth factor/scatter factor (HGF/SF) plays critical roles in cancer progression through its specific receptor, MET. HGF/SF is usually synthesized and secreted as an inactive proform (pro-HGF/SF) by stromal cells, such as fibroblasts. Several serine proteases are reported to convert pro-HGF/SF to mature HGF/SF and among these, HGF activator (HGFA) and matriptase are the most potent activators. Increased activities of both proteases have been observed in various cancers. HGFA is synthesized mainly by the liver and secreted as an inactive pro-form. In cancer tissues, pro-HGFA is likely activated by thrombin and/or human kallikrein 1-related peptidase (KLK)-4 and KLK-5. Matriptase is a type II transmembrane serine protease that is expressed by most epithelial cells and is also synthesized as an inactive zymogen. Matriptase activation is likely to be mediated by autoactivation or by other trypsin-like proteases. Recent studies revealed that matriptase autoactivation is promoted by an acidic environment. Given the mildly acidic extracellular environment of solid tumors, matriptase activation may, thus, be accelerated in the tumor microenvironment. HGFA and matriptase activities are regulated by HGFA inhibitor (HAI)-1 (HAI-1) and/or HAI-2 in the pericellular microenvironment. HAIs may have an important role in cancer cell biology by regulating HGF/SF-activating proteases.

Hepatocyte growth factor activator (HGFA) is a serine protease initially identified as a potent activator of hepatocyte growth factor/scatter factor. Hepatocyte growth factor/scatter factor is known to be critically involved in tissue morphogenesis, regeneration, and tumor progression, via its receptor, MET. In vivo, HGFA also activates macrophage‐stimulating protein, which has roles in macrophage recruitment and inflammatory processes, cellular survival and wound healing through its receptor, RON. Therefore, the pericellular activity of HGFA might be an important factor regulating the activities of these multifunctional cytokines in vivo. HGFA is secreted mainly by the liver, circulates in the plasma as a zymogen (pro‐HGFA), and is activated in response to tissue injury, including tumor growth. In addition, local production of pro‐HGFA by epithelial, stromal or tumor cells has been reported. Although the generation of HGFA‐knockout mice revealed that the role played by HGFA in normal development and physiological settings can be compensated for by other protease systems, HGFA has important roles in regeneration and initial macrophage recruitment in injured tissue in vivo. Insufficient activity of HGFA results in impaired regeneration of severely damaged mucosal epithelium, and may contribute to the progression of fibrotic lung diseases. On the other hand, deregulated excess activity of HGFA may be involved in the progression of some types of cancer.

A loss of balance between cell membrane-associated proteases and their inhibitors may underlie cancer invasion and metastasis. We analysed the roles of a membrane- associated serine protease inhibitor, HAI-1, in oral squamous cell carcinoma (OSCC). While membranous HAI-1 was widely observed in cancer cells of human OSCC tissues, this was significantly reduced at the infiltrative invasion front. In vitro, HAI-1 was detected in all eight OSCC cell lines examined, in which its cognate membrane protease, matriptase was also expressed. HAI-1 expression knock-down (KD) in OSCC lines, SAS and HSC-3, reduced the growth of both lines in vitro but significantly enhanced SAS tumourigenicity in vivo, which was accompanied by histological changes suggestive of the epithelial-mesenchymal transition. Both HAI-1-KD lines also exhibited significantly enhanced migratory capability, and membrane-associated but not truncated HAI-1 was required to rescue this phenotype. Other OSCC lines (HSC-2, Sa3, Ca9-22) also showed enhanced migration in response to HAI-1 KD. The enhanced migration is partly attributed to dysregulation of matriptase, as simultaneous matriptase KD alleviated the migration of HAI-1-KD cells. HAI-1 deficiency also altered the expression of CD24, S100A4, CCND2 and DUSP6, all of which are involved in tumour progression. While matriptase was involved in the increased CD24 expression associated with HAI-1 deficiency, the protease appeared to be not responsible for the altered expression of other genes. Therefore, a matriptase-independent mechanism for the invasiveness associated with HAI-1 KD is also present. Together, these observations suggest that HAI-1 has a crucial suppressive role in OSCC cell invasiveness.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1/SPINT1) is a membrane-bound serine protease inhibitor expressed on the surface of epithelial cells. Although HAI-1/SPINT1 is abundantly expressed in the intestinal epithelium, its role in intestinal tumorigenesis is not known. In this study, we investigated the role of Hai-1/Spint1 in intestinal tumorigenesis using mouse models. The membranous Hai-1/Spint1 immunoreactivity was decreased in murine ApcMin/þ tumors and also in carcinogen (azoxymethane treatment followed by dextran sodium sulfate administration)-induced colon tumors compared with the adjacent non-neoplastic epithelium. The decreased immunoreactivity appeared to be due to sheddase activity of membrane-type 1 matrix metalloprotease. Then, we examined the effect of intestine-specific deletion of Spint1 gene on Apc Min/þ mice. The loss ofHai-1/Spint1 significantly accelerated tumor formation in Apc Min/þ mice and shortened their survival periods.Activation of HGF was enhanced in Hai-1/Spint1-deficient Apc Min/þ intestine. Gene expression profiling revealed upregulation of the Wnt/b-catenin signaling circuit, claudin-2 expression, and angiogenesis not only in tumor tissue but also in the background mucosa without macroscopic tumors in Hai-1/Spint1-deficient Apcintestine. Intestinal deletion of Spint1 also enhanced the susceptibility to carcinogen-induced colon tumorigenicity of wild-type Apc mice. Our findings suggest that HAI-1/SPINT1 has a crucial role in suppressing intestinal tumorigenesis, which implies a novel link between epithelial cell surface serine protease inhibitors and protection from carcinogenic stimuli. Cancer Res; 73(8); 2659-70. Ó2013 AACR.

Thymic stromal lymphopoietin (TSLP) activates dendritic cells to induce Th2-mediated inflammation. Periostin, an extracellular matrix protein produced by fibroblasts, induces chronic inflammation by stimulating TSLP production. Recently, a reinforcing cycle linking Th2-type immune responses with periostin-induced keratinocyte activation has been proposed in atopic dermatitis pathogenesis. In this study, we investigated the role of TSLP and periostin in the development of cutaneous T-cell lymphoma (CTCL), where Th2 cytokines and chemokines are also dominant. TSLP and periostin mRNA expression levels were elevated in CTCL lesional skin, both of which correlated with IL4 expression levels. In vitro and ex vivo, IL4 or IL13 stimulated periostin expression by dermal fibroblasts, and fibroblasts from CTCL lesional skin expressed higher levels of periostin than those from control skin. Serum periostin levels of CTCL patients were also significantly higher than those of healthy individuals. Hut78 and MJ, CTCL cell lines, and peripheral blood mononuclear cells from leukemic CTCL patients expressed the TSLP receptor. TSLP induced production of IL4 and IL13 by Hut78 and MJ cells through the activation of STAT5. Moreover, TSLP induced proliferation of CTCL cells both in vitro and in vivo These data suggest that periostin-mediated TSLP production by keratinocytes directly stimulates CTCL tumor cell growth in addition to inducing a Th2-dominant tumor environment in CTCL. Cancer Res; 76(21); 6241-52. ©2016 AACR.

The lineage commitment of CD4+ T cells is coordinately regulated by signals through the T cell receptor and cytokine receptors, yet how these signals are integrated remains elusive. Here we find that mice lacking Dock2, a Rac activator in lymphocytes, developed allergic disease through a mechanism dependent on CD4+ T cells and the interleukin 4 receptor (IL-4R). Dock2-deficient CD4+ T cells showed impaired antigen-driven downregulation of IL-4Ralpha surface expression, resulting in sustained IL-4R signaling and excessive T helper type 2 responses. Dock2 was required for T cell receptor-mediated phosphorylation of the microtubule-destabilizing protein stathmin and for lysosomal trafficking and the degradation of IL-4Ralpha. Thus, Dock2 links T cell receptor signals to downregulation of IL-4Ralpha to control the lineage commitment of CD4+ T cells.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/ Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1؉/؉ blastocysts with Hai-1/Spint1 ؊/؊ embryonic stem cells successfully generated high-chimeric Hai-1/Spint1 ؊/؊ embryos (B6Hai-1 ؊/؊High ) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1 ؊/؊ mice was caused by placental dysfunction. However, newborn B6Hai-1 ؊/؊High mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.