Type 2 3a-hydroxysteroid dehydrogenase (3a-HSD) is a multi-functional enzyme that possesses 3a-, 17b-and 20a-HSD, as well as prostaglandin (PG) F synthase activities and catalyzes androgen, estrogen, progestin and PG metabolism. Type 2 3a-HSD was cloned from human prostate, is a member of the aldo-keto reductase (AKR) superfamily and was named AKR1C3. In androgen target tissues such as the prostate, AKR1C3 catalyzes the conversion of D 4 -androstene-3,17-dione to testosterone, 5a-dihydrotestosterone to 5a-androstane-3a,17b-diol (3a-diol), and 3a-diol to androsterone. Thus AKR1C3 may regulate the balance of androgens and hence transactivation of the androgen receptor in these tissues. Tissue distribution studies indicate that AKR1C3 transcripts are highly expressed in human prostate. To measure AKR1C3 protein expression and its distribution in the prostate, we raised a monoclonal antibody specifically recognizing AKR1C3. This antibody allowed us to distinguish AKR1C3 from other AKR1C family members in human tissues. Immunoblot analysis showed that this monoclonal antibody binds to one species of protein in primary cultures of prostate epithelial cells and in LNCaP prostate cancer cells. Immunohistochemistry with this antibody on human prostate detected strong nuclear immunoreactivity in normal stromal and smooth muscle cells, perineurial cells, urothelial (transitional) cells, and endothelial cells. Normal prostate epithelial cells were only faintly immunoreactive or negative. Positive immunoreactivity was demonstrated in primary prostatic adenocarcinoma in 9 of 11 cases. Variable increases in immunoreactivity for AKR1C3 was also demonstrated in non-neoplastic changes in the prostate including chronic inflammation, atrophy and urothelial (transitional) cell metaplasia. We conclude that elevated expression of AKR1C3 is highly associated with prostate carcinoma. Although the biological significance of elevated AKR1C3 in prostatic carcinoma is uncertain, AKR1C3 may be responsible for the trophic effects of androgens and/or PGs on prostatic epithelial cells.

Aims Proprotein convertase subtilisin/kexin type-9 (PCSK9), a molecular determinant of low-density lipoprotein (LDL) receptor (LDLR) fate, has emerged as a promising therapeutic target for atherosclerotic cardiovascular diseases. However, the precise mechanism by which PCSK9 regulates the internalization and lysosomal degradation of LDLR is unknown. Recently, we identified adenylyl cyclase-associated protein 1 (CAP1) as a receptor for human resistin whose globular C-terminus is structurally similar to the C-terminal cysteine-rich domain (CRD) of PCSK9. Herein, we investigated the role of CAP1 in PCSK9-mediated lysosomal degradation of LDLR and plasma LDL cholesterol (LDL-C) levels. Methods and results The direct binding between PCSK9 and CAP1 was confirmed by immunoprecipitation assay, far-western blot, biomolecular fluorescence complementation, and surface plasmon resonance assay. Fine mapping revealed that the CRD of PCSK9 binds with the Src homology 3 binding domain (SH3BD) of CAP1. Two loss-of-function polymorphisms found in human PCSK9 (S668R and G670E in CRD) were attributed to a defective interaction with CAP1. siRNA against CAP1 reduced the PCSK9-mediated degradation of LDLR in vitro. We generated CAP1 knock-out mice and found that the viable heterozygous CAP1 knock-out mice had higher protein levels of LDLR and lower LDL-C levels in the liver and plasma, respectively, than the control mice. Mechanistic analysis revealed that PCSK9-induced endocytosis and lysosomal degradation of LDLR were mediated by caveolin but not by clathrin, and they were dependent on binding between CAP1 and caveolin-1. Conclusion We identified CAP1 as a new binding partner of PCSK9 and a key mediator of caveolae-dependent endocytosis and lysosomal degradation of LDLR.

Immune escape of a tumor from tumor-infiltrating lymphocytes (TILs) is induced by PD-L1, which is suppressed by miR-197. We investigated the clinicopathologic implications of the miR-197/PD-L1 axis and its effects on TILs and the clinicopathologic features of oral squamous cell carcinoma (OSCC). We used RT-PCR and immunohistochemistry in 68 OSCC patients to analyze the correlations between tumoral expression of miR-197 and PD-L1 and the degree of tumoral invasion by TILs (CD3+, CD4+, CD8+, PD-1+, FoxP3+, and CD20+ lymphocytes). PD-L1 levels correlated inversely with miR-197 but correlated positively with TILs. The aggressive features of OSCC, including high stage, angiolymphatic invasion, perineural invasion, and death, were associated with TIL depletion. High T stage (T4) tumors also had low PD-L1 but had high miR-197 expression. In a univariate survival analysis of the full cohort, high miR-197 was associated with poor overall survival, whereas high PD-L1 expression (2+) associated with good overall survival. In a multivariate analysis stratified based on miR-197 (median), high PD-L1 expression (2+) was an independent favorable prognostic factor for overall survival (P = 0.040) in the miR-197high subgroup but not the miR-197low subgroup. These findings may have clinicopathologic implications for the miR-197/PD-L1 axis and TILs in OSCC.

Hypoxic microenvironment plays an important role in determining stem cell fates. However, it is controversial to which direction between self-renewal and differentiation the hypoxia drives the stem cells. Here, we investigated whether a short exposure to hypoxia (termed ‘hypoxic-priming’) efficiently directed and promoted mouse embryonic stem cells (mESCs) to differentiate into vascular-lineage. During spontaneous differentiation of embryoid bodies (EBs), hypoxic region was observed inside EB spheroids even under normoxic conditions. Indeed, hypoxia-primed EBs more efficiently differentiated into cells of vascular-lineage, than normoxic EBs did. We found that hypoxia suppressed Oct4 expression via direct binding of HIF-1 to reverse hypoxia-responsive elements (rHREs) in the Oct4 promoter. Furthermore, vascular endothelial growth factor (VEGF) was highly upregulated in hypoxia-primed EBs, which differentiated towards endothelial cells in the absence of exogenous VEGF. Interestingly, this differentiation was abolished by the HIF-1 or VEGF blocking. In vivo transplantation of hypoxia-primed EBs into mice ischemic limb elicited enhanced vessel differentiation. Collectively, our findings identify that hypoxia enhanced ESC differentiation by HIF-1-mediated inverse regulation of Oct4 and VEGF, which is a novel pathway to promote vascular-lineage differentiation.