Laminin-5, a major adhesive ligand for epithelial cells, undergoes processing of its ␥2 and ␣3 chains. This study investigated the mechanism of laminin-5 processing by keratinocytes. BI-1 (BMP-1 isoenzyme inhibitor-1), a selective inhibitor of a small group of astacin-like metalloproteinases, which includes bone morphogenetic protein 1 (BMP-1), mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mammalian Tolloid-like 2 (mTLL-2), inhibited the processing of laminin-5 ␥2 and ␣3 chains in keratinocyte cultures in a dose-dependent manner. In a proteinase survey, all BMP-1 isoenzymes processed human laminin-5 ␥2 and ␣3 chains to 105-and 165-kDa fragments, respectively. In contrast, MT1-MMP and MMP-2 did not cleave the ␥2 chain of human laminin-5 but processed the rat laminin ␥2 chain to an 80-kDa fragment. An immunoblot and quantitative PCR survey of the BMP-1 isoenzymes revealed expression of mTLD in primary keratinocyte cultures but little or no expression of BMP-1, mTLL-1, or mTLL-2. mTLD was shown to cleave the ␥2 chain at the same site as the previously identified BMP-1 cleavage site. In addition, mTLD/BMP-1 null mice were shown to have deficient laminin-5 processing. Together, these data identify laminin-5 as a substrate for mTLD, suggesting a role for laminin-5 processing by mTLD in the skin.Proteolysis of the extracellular matrix is emerging as a key mechanism in processes such as wound healing and tumor metastasis (1, 2). Although most studies have investigated the role of serine proteases and matrix metalloproteases, members of the astacin and ADAM (a disintegrin and metalloprotease) families have also been implicated in this process (1, 2). Laminin-5, the major component of epithelial basement membranes, is a heterotrimeric protein consisting of ␣3, ␤3, and ␥2 subunits (3, 4). Laminin-5 undergoes extracellular proteolysis of the ␣3 chain from a 200-to a 165-kDa form and of the ␥2 chain from a 155-to a 105-kDa form (5). Through its interaction with ␣ 3 ␤ 1 (6, 7), ␣ 6 ␤ 4 (8), and ␣ 2 ␤ 1 integrins (9), laminin-5 supports epithelial cell adhesion (3, 10), and migration (11, 12).Several proteases have been implicated in laminin-5 processing. Exogenous addition of matrix metalloprotease 2 (MMP-2) 1 cleaved the ␥2 subunit of rat laminin-5 (12). A subsequent study suggested that membrane type 1 matrix metalloprotease (MT1-MMP) may play a role in cleaving laminin-5 (13). Cleavage of laminin-5 by plasmin converted the ␣3 chain into the 165-kDa form observed in human breast and rat epithelial cells and capable of nucleating hemidesmosomes (14). Bone morphogenetic protein 1 (BMP-1) has also been implicated in laminin-5 proteolysis. N-terminal sequencing of the 105-kDa ␥2 chain obtained from human keratinocytes revealed a cleavage site that matched the minimal consensus sequence of this metalloprotease (15). In vitro studies demonstrated that BMP-1 cleaved the recombinant ␥2 short arm at the predicted site and that the enzyme cleaved both the ␣3 and ␥2 chains of whole laminin-5 to generate characterist...

17 beta-Hydroxysteroid dehydrogenases/17-ketosteroid reductases (17HSDs) modulate the biological activity of certain estrogens and androgens by catalyzing reductase or dehydrogenase reactions between 17-keto- and 17 beta-hydroxysteroids. In the present study, we demonstrate expression cloning of a novel type of 17HSD, chronologically named 17HSD type 7, from the HC11 cell line derived from mouse mammary gland. The cloned cDNA, 1.7 kb in size, encodes a protein of 334 amino acids with a calculated molecular mass of 37,317 Da. The primary structure contains segments characteristic of enzymes belonging to the short-chain dehydrogenase/reductase superfamily. Strikingly, mouse 17HSD type 7 (m17HSD7) shows 89% identity with a recently cloned rat protein called PRL receptor-associated protein (PRAP). The function of PRAP has not yet been demonstrated. The enzymatic characteristics of m17HSD7 and RT-PCR-cloned rat PRAP (rPRAP) were analyzed in cultured HEK-293 cells, where both of the enzymes efficiently catalyzed conversion of estrone (E1) to estradiol (E2). With other substrates tested no detectable 17HSD or 20 alpha-hydroxysteroid dehydrogenase activities were found. Kinetic parameters for m17HSD7 further indicate that E1 is a preferred substrate for this enzyme. Relative catalytic efficiencies (Vmax/K(m) values) for E1 and E2 are 244 and 48, respectively. As it is the case with rPRAP, m17HSD7 is most abundantly expressed in the ovaries of pregnant animals. Further studies show that the rat enzyme is primarily expressed in the middle and second half of pregnancy, in parallel with E2 secretion from the corpus luteum. The mRNA for m17HSD7 is also apparent in the placenta, and a slight signal for m17HSD7 is found in the ovaries of adult nonpregnant mice, in the mammary gland, liver, kidney, and testis. Altogether, because of their similar primary structures, enzymatic characteristics, and the tissue distribution of m17HSD7 and rPRAP, we suggest that rPRAP is rat 17HSD type 7. Furthermore, the results indicate that 17HSD7 is an enzyme of E2 biosynthesis, which is predominantly expressed in the corpus luteum of the pregnant animal.

The biological activity of certain estrogens and androgens is modulated by enzymes called 17p-hydroxysteroid dehydrogenases (I~P-HSDS), which catalyze the interconversion between less active 17-oxosteroid and more active 17P-hydroxysteroid forms. In the present report, we describe cloning of mouse 17P-HSD type-I cDNA from an ovarian library generated from 4,4'-( 1,2-diethyl-I ,2-ethenediyl)bisphenol-(diethylstiIbestro1)-treated mice, and characterization of the corresponding enzyme. The open reading frame of the mouse 17P-HSD type-1 cDNA encodes a peptide of 344 amino acid residues with a predicted molecular mass of 3678.5 Da. The mouse 17P-HSD type-1 enzyme shares 63 % and 93 % overall identity with human and rat 17P-HSD type-I enzymes, respectively, and the most striking differences between the mouse and human type-1 enzymes are between the amino acid residues 197 and 230 and in the carboxy terminus of the enzymes. Similarly to the human 17P-HSD type-I enzyme, the mouse type-I enzyme primarily catalyzes reductive reactions from 17-0x0 forms to 17P-hydroxy forms in intact cultured cells, but unlike the human type-I enzyme, the mouse enzyme does not prefer phenolic over neutral substrates. Thus, mouse 17P-HSD type 1 catalyzes reduction of androst-4-ene-3,17-dione (androstenedione) to 17P-hydroxyandrost-4-en-3-one (testosterone) as efficiently as 3P-hydroxyestra-l,3,.5(10)-trien-17-one (estrone) to estra-1,3,5(10)-triene-3~,17~-diol (estradiol). 17P-HSD type 1 is predominantly expressed in mouse ovaries, in which it is located in granulosa cells.Keywords: 17-hydroxysteroid dehydrogenases ; cloning ; mouse; estrogen ; B1 repetitive sequence.17P-Hydroxysteroid dehydrogenases (17p-HSDs) catalyze the conversion of neutral and phenolic 17-oxosteroids to 17P-hydroxysteroids and vice versa. Both androgens and estrogens are biologically more active in their l7P-hydroxy configurations, such as 17P-hydroxyandrost-4-en-3-one (testosterone) and estra-1,3,S(10)-triene-3~,17~-diol (estradiol), than in their 17-0x0 configuration. Thus, 17P-HSDs, along with other steroidogenic enzymes such as 3P-hydroxysteroid dehydrogenases and Sa-reductases, modulate the biological activity of the sex steroids.

Laminin-332 is critical for squamous cell carcinoma (SCC) tumorigenesis, but targeting it for cancer therapy has been unachievable due to key role of laminin-332 in promoting tissue integrity. Here, we show that a portion of laminin-332, termed G45, which is proteolytically removed and absent in normal tissues, is prominently expressed in most human SCC tumors and plays an important role in human SCC tumorigenesis. Primary human keratinocytes lacking G45 (#G45) showed alterations of basal receptor organization, impaired matrix deposition, and increased migration. After SCC transformation, the absence of G45 domain in #G45 cells was associated with deficient extracellular signalregulated kinase and phosphotidylinositol 3-kinase (PI3K) pathway activation, impaired invasion, deficient metalloproteinase activity, and absent tumorgenicity in vivo. Expression of G45 or activated PI3K subunit in #G45 cells reversed these abnormalities. G45 antibody treatment induced SCC tumor apoptosis, decreased SCC tumor proliferation, and markedly impaired human SCC tumorigenesis in vivo without affecting normal tissue adhesion. These results show a remarkable selectivity of expression and function for laminin-332 G45 in human SCC tumorigenesis and implicate it as a specific target for anticancer therapy. [Cancer Res 2008;68(8):2885-94]