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Objectives-Characterize the expression and glucuronidation activities of the human UDPglucuronosyltransferase (UGT) 2A2.Methods-UGT2A1 was cloned from nasal mucosa mRNA. Synthetic cDNA for UGT2A2 was constructed assuming exon sharing between UGT2A1 and UGT2A2 (Mackenzie et al., Pharmacogenetics and Genomics 2005, 15:677-685). Exon 1 of UGT2A2 was amplified from genomic DNA and combined with exons 2-6 of UGT2A1. UGT2A3 was cloned from liver mRNA. Quantitative RT-PCR was used to evaluate the expression of all the three UGTs of subfamily 2A in different tissues. Recombinant UGT2A1, UGT2A2 and UGT2A3 were expressed in baculovirus-infected insect cells and analyzed for glucuronidation activity towards different substrates.Results-DNA sequencing of reverse-transcribed PCR (RT-PCR) products from human nasal mucosa mRNA, confirmed exon sharing between UGT2A1 and UGT2A2. In addition, it indicated that the N-terminal signal peptide sequence of UGT2A2 is the longest among the human UGTs. Quantitative RT-PCR revealed that both UGT2A1 and UGT2A2 are mainly expressed in the nasal mucosa, and that their expression level in fetal samples was much higher than in adults. Activity assays with recombinant UGTs 2A1-2A3 demonstrated broad substrate selectivity for UGT2A1 and UGT2A2. While glucuronidation rates and substrate affinities were mostly higher in UGT2A1, the K m values for UDP-glucuronic acid were similar in both UGTs. In addition, there were regioselectivity differences between the two UGTs and, with a few substrates, particularly ethinylestradiol, the activity of UGT2A2 was higher.Conclusions-UGT2A2 is mainly expressed in the nasal mucosa and it has glucuronidation activity towards several different endo-and xenobiotic substrates.

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Summary The SCARECROW (SCR) gene is central to root radial patterning. Its expression has not been investigated in conifers with morphologically different root types. Additional interest in SCR functions in the Pinus sylvestris root system comes from the effect of ectomycorrhiza formation on the short root apical structure. Here, the P. sylvestris SCR gene (PsySCR) was cloned and its expression investigated by northern blot and in situ hybridization of primary, lateral and short roots and mycorrhiza. Short root dichotomization was induced by auxin transport inhibitor (N‐1‐naphthylphthalamic acid (NPA)). PsySCR has conserved GRAS family protein motifs at the C‐terminus and a variable N‐terminus. PsySCR expression occurred in young root tissue and mycorrhiza. In root sections the PsySCR signal runs through the tip in initials for stele and root cap column and becomes upwards‐restricted to endodermis in all root types. The PsySCR expression pattern suggests for the first time a regulatory role for SCR in maintaining the endodermal characteristics and radial patterning of roots with open meristem organization. The specific PsySCR localization is also an excellent marker for investigation of the dichotomization process in short roots.

While the role of LKB1 mutations in the Peutz-Jeghers polyposis syndrome (PJS) is uncontroversial, the originating cell type remains unclear as Lkb1 mutations in both epithelial cells and stromal smooth muscle cells (SMCs) have been proposed as tumor drivers. Since SMCs do not represent a major fraction of stromal cells in polyps, altered signaling from Lkb1-deficient SMCs to epithelium has been suggested as a possible mechanism for polyposis. Here we investigate the cell type origin and tumor development mechanism of PJS type polyps in mice by targeting Lkb1 in either early mesenchymal progenitors (Twist2-Cre) or in stromal fibroblasts (Fsp1-Cre). Remarkably, both Twist2 (Twist2-Cre;Lkb1flox/+) and Fsp1-driven (Fsp1-Cre;Lkb1flox/+ and Fsp1-Cre;Lkb1flox/flox) Lkb1 deletion led to formation of PJS-type gastrointestinal polyps predominantly in the stomach, as noted before for Lkb1+/- mice. Furthermore, lineage-tracing experiments demonstrated that Lkb1 deletion results in early local expansion of Lkb1-deficient myofibroblast-like alpha smooth muscle actin-expressing cells between gastric glands, and subsequent clonal expansion filling the stroma of the forming polyps. The simultaneous expansion of the adjacent epithelium seems to be secondary to the stromal growth, based on lack of noticeable genetic alterations in the epithelial cells. Immunohistochemical and mRNA analyses demonstrate that the polyps arising from the stromal deletion models are indistinguishable from Lkb1+/- mice and PJS patient polyps. These results indicate that polyps in the Twist2-Cre and Fsp1-Cre mice represent stromal tumors, and suggest that the identified tumorigenic mechanism is shared in PJS syndrome. Citation Format: Saara Ollila, Kaisa Laajanen, Iris Wong, Kari Vaahtomeri, Tomi P. Mäkelä. Clonal expansion of Lkb1-deficient stromal cells underlies polyp development in mouse models of Peutz-Jeghers syndrome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4857. doi:10.1158/1538-7445.AM2014-4857