Regulation of the Matriptase-Prostasin Cell Surface Proteolytic Cascade by Hepatocyte Growth Factor Activator Inhibitor-1 during Epidermal Differentiation

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates

Suppression of AP1 Transcription Factor Function in Keratinocyte Suppresses Differentiation

Our previous study shows that inhibiting activator protein one (AP1) transcription factor function in murine epidermis, using dominant-negative c-jun (TAM67), increases cell proliferation and delays differentiation. To understand the mechanism of action, we compare TAM67 impact in mouse epidermis and in cultured normal human keratinocytes. We show that TAM67 localizes in the nucleus where it forms TAM67 homodimers that competitively interact with AP1 transcription factor DNA binding sites to reduce endogenous jun and fos factor binding. Involucrin is a marker of keratinocyte differentiation that is expressed in the suprabasal epidermis and this expression requires AP1 factor interaction at the AP1-5 site in the promoter. TAM67 interacts competitively at this site to reduce involucrin expression. TAM67 also reduces endogenous c-jun, junB and junD mRNA and protein level. Studies with c-jun promoter suggest that this is due to reduced transcription of the c-jun gene. We propose that TAM67 suppresses keratinocyte differentiation by interfering with endogenous AP1 factor binding to regulator elements in differentiation-associated target genes, and by reducing endogenous c-jun factor expression

TIG3 Tumor Suppressor-Dependent Organelle Redistribution and Apoptosis in Skin Cancer Cells

TIG3 is a tumor suppressor protein that limits keratinocyte survival during normal differentiation. It is also important in cancer, as TIG3 level is reduced in tumors and in skin cancer cell lines, suggesting that loss of expression may be required for cancer cell survival. An important goal is identifying how TIG3 limits cell survival. In the present study we show that TIG3 expression in epidermal squamous cell carcinoma SCC-13 cells reduces cell proliferation and promotes morphological and biochemical apoptosis. To identify the mechanism that drives these changes, we demonstrate that TIG3 localizes near the centrosome and that pericentrosomal accumulation of TIG3 alters microtubule and microfilament organization and organelle distribution. Organelle accumulation at the centrosome is a hallmark of apoptosis and we demonstrate that TIG3 promotes pericentrosomal organelle accumulation. These changes are associated with reduced cyclin D1, cyclin E and cyclin A, and increased p21 level. In addition, Bax level is increased and Bcl-XL level is reduced, and cleavage of procaspase 3, procaspase 9 and PARP is enhanced. We propose that pericentrosomal localization of TIG3 is a key event that results in microtubule and microfilament redistribution and pericentrosomal organelle clustering and that leads to cancer cell apoptosis

Embryonic AP1 Transcription Factor Deficiency Causes a Collodion Baby-Like Phenotype

AP1 transcription factors are important controllers of gene expression in the epidermis, and altered AP1 factor function can perturb keratinocyte proliferation and differentiation. However, our understanding of how AP1 signaling changes may underlie or exacerbate skin disease is limited. We have shown that inhibiting AP1 factor function in suprabasal adult epidermis leads to reduced filaggrin levels and to a phenotype that resembles the genetic disorder ichthyosis vulgaris. We now show that inhibiting AP1 factor function during development in embryonic epidermis produces marked phenotypic changes including reduced filaggrin mRNA and protein levels, compromised barrier function, marked ultrastructural change, and enhanced dehydration susceptibility that resembles the phenotype observed in the flaky tail mouse, a model for ichthyosis vulgaris. In addition, the AP1 factor-deficient newborn mice display a collodion membrane phenotype that is not observed in flaky tail mice or in newborn individuals with ichthyosis vulgaris but is present in other forms of ichthyosis. This mixed phenotype suggests the need for a better understanding of the possible role of filaggrin loss and AP1 transcription factor deficiency in ichthyoses and collodion membrane formation

TIG3 reduces cell cycle progression.

<p><b>A</b> SCC-13 cells grown on coverslips were infected with 10 MOI of EV or TIG3-encoding virus and after 24 h treated with 10 µM BrdU for 2 h and then fixed and stained with anti-BrdU (red) and anti-TIG3 (green). BrdU incorporation is a marker of the synthesis phase of the cell cycle. The number of BrdU positive cells as a percentage of total cell number is presented beneath each panel. The values are mean ± SEM (n = 3) and the values are significantly different as determined by Student's t-test (p<0.001). Bars = 10 µm. <b>B</b> SCC-13 cells were collected for flow cytometry at 24 h post-infection with EV or TIG3-encoding virus. Cells were stained with 50 µg/ml propidium iodide prior to analysis. TIG3 reduces events in G1 and increases subG1 events. <b>C</b> At 24 h post-infection, cells were harvested and extracts prepared for detection of cell cycle regulatory proteins. Molecular weights are indicated to the left of the blot in kDa. <b>D</b> Cells were treated as above and then harvested for detection of p21 encoding mRNA by real time-PCR. A similar result was observed in each of three experiments.</p

TIG3 induces apoptosis.

<p><b>A</b> SCC-13 cells were infected with 10 MOI of EV or TIG3-encoding virus and after 24 h lysates were prepared for detection of apoptosis markers. The bracket indicates high molecular weight crosslinked TIG3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023230#pone.0023230-Sturniolo1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023230#pone.0023230-Sturniolo2" target="_blank">[11]</a>. Molecular weights are indicated to the left of the blot in kDa. <b>B</b> At 24 h post-infection SCC-13 cells were fixed and stained with anti-cleaved PARP (red). TIG3 increases cleaved PARP staining. The percentage of cleaved PARP positive cells is presented in each panel (mean ± SD). The arrows indicate cleaved PARP-positive cells. Similar results were observed in each of three experiments.</p

TIG3 decreases cell survival.

<p>Subconfluent cultures of SCC-13 cells, growing in 3.8 cm² wells, were infected with 10 MOI tAd5-EV or tAd5-TIG3. At 0, 24, 48, and 72 h post-infection, cells were counted and lysates prepared. <b>A</b> TIG3 expression decreases cell number. Values are mean ± SEM, n = 3. Those comparisons marked by an asterisk are significantly different as determined by Student's t-test (p<0.001). <b>B</b> TIG3 is detected by immunoblot in tAd5-TIG3 infected SCC-13 cells. The monomer is visible at 18 kDa and the bracket indicates higher molecular weight crosslinked TIG3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023230#pone.0023230-Sturniolo1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023230#pone.0023230-Sturniolo2" target="_blank">[11]</a>. Molecular weights are indicated to the left of the blot in kDa.</p

TIG3 impact on organelle marker protein level.

<p>SCC-13 cells were infected by 10 MOI tAd5-EV or tAd5-TIG3 and after 24 h cell lysates were prepared for immunoblot detection of the indicated proteins. TIG3 expression reduces the level of rab11, GM130 and EEA1, but does not alter LAMP1 level. Molecular weights are indicated to the left of the blot in kDa.</p