Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation.

We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9(+/-);bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9(+/-);bt/+ and bt/bt mice, demonstrating cooperation of these genes. Adamts20 is expressed in palatal mesenchyme, whereas Adamts9 is expressed exclusively in palate microvascular endothelium. Palatal shelves isolated from Adamts9(+/-);bt/bt mice fused in culture, suggesting an intact epithelial TGFß3 signaling pathway. Cleft palate resulted from a temporally specific delay in palatal shelf elevation and growth towards the midline. Mesenchyme of Adamts9(+/-);bt/bt palatal shelves had reduced cell proliferation, a lower cell density and decreased processing of versican (VCAN), an extracellular matrix (ECM) proteoglycan and ADAMTS9/20 substrate, from E13.5 to E14.5. Vcan haploinsufficiency led to greater penetrance of cleft palate in bt mice, with a similar defect in palatal shelf extension as Adamts9(+/-);bt/bt mice. Cell density was normal in bt/bt;Vcan(hdf)(/+) mice, consistent with reduced total intact versican in ECM, but impaired proliferation persisted in palate mesenchyme, suggesting that ADAMTS-cleaved versican is required for cell proliferation. These findings support a model in which cooperative versican proteolysis by ADAMTS9 in vascular endothelium and by ADAMTS20 in palate mesenchyme drives palatal shelf sculpting and extension.

ADAMTSL3/punctin-2, a gene frequently mutated in colorectal tumors, is widely expressed in normal and malignant epithelial cells, vascular endothelial cells and other cell types, and its mRNA is reduced in colon cancer.

ADAMTSL3/punctin-2 is a secreted glycoprotein that resembles the ADAMTS proteases. Recently, identification of frequent ADAMTSL3 mutations in colorectal cancer suggested it might have a regulatory role in cellular homeostasis in colorectal epithelium or in pathways to colorectal malignancy. Here, we used in situ hybridization to validate ADAMTSL3 antibodies for IHC of a variety of normal and malignant tissues, including colon cancer. Quantitative real-time PCR (RTQ-PCR) was used to compare mRNA expression levels in colon carcinoma (n = 10) and adjacent normal colon. ADAMTSL3 is expressed in epithelial cells of the colon, fallopian tube, skin, breast, prostate, epididymis, liver, pancreatic islets and bile ducts, as well as by vascular endothelial cells, smooth muscle cells, fibroblasts, cortical and ganglionic neurons and cardiac myocytes. Malignant epithelial cells in colon cancer, as well as breast, prostate, renal and skin tumors expressed ADAMTSL3. Normal colon showed stronger immunostaining of surface than basal crypt epithelium and staining of a variety of cells within the lamina propria and submucosa. Colon carcinomas demonstrated weaker staining in tumor cells than normal colon epithelium and weak stromal staining. RTQ-PCR comparison of ADAMTSL3 mRNA in colon carcinoma and adjacent normal colon demonstrated a statistically significant reduction in the tumors, possibly reflecting their decreased stromal content and lack of complete differentiation of tumor samples. The major findings of these studies are that ADAMTSL3 is expressed in numerous tissues, suggesting a broader regulatory role than in colorectal epithelium alone, and that colorectal cancer has both structural mutations as well as decreased expression of ADAMTSL3.