Inhibition of phosphodiesterase-4 (PDE4) activity triggers luminal apoptosis and AKT dephosphorylation in a 3-D colonic-crypt model.

BACKGROUND: We previously established a three-dimensional (3-D) colonic crypt model using HKe3 cells which are human colorectal cancer (CRC) HCT116 cells with a disruption in oncogenic KRAS, and revealed the crucial roles of oncogenic KRAS both in inhibition of apoptosis and in disruption of cell polarity; however, the molecular mechanism of KRAS-induced these 3-D specific biological changes remains to be elucidated. RESULTS: Among the genes that were upregulated by oncogenic KRAS in this model, we focused on the phosphodiesterase 4B (PDE4B) of which expression levels were found to be higher in clinical tumor samples from CRC patients in comparison to those from healthy control in the public datasets of gene expression analysis. PDE4B2 was specifically overexpressed among other PDE4 isoforms, and re-expression of oncogenic KRAS in HKe3 cells resulted in PDE4B overexpression. Furthermore, the inhibition of PDE4 catalytic activity using rolipram reverted the disorganization of HCT116 cells into the normal physiologic state of the epithelial cell polarity by inducing the apical assembly of ZO-1 (a tight junction marker) and E-cadherin (an adherens junction marker) and by increasing the activity of caspase-3 (an apoptosis marker) in luminal cavities. Notably, rolipram reduced the AKT phosphorylation, which is known to be associated with the disruption of luminal cavity formation and CRC development. Similar results were also obtained using PDE4B2-shRNAs. In addition, increased expression of PDE4B mRNA was found to be correlated with relapsed CRC in a public datasets of gene expression analysis. CONCLUSIONS: These results collectively suggested that PDE4B is upregulated by oncogenic KRAS, and also that the inhibition of PDE4 catalytic activity can induce both epithelial cell polarity and luminal apoptosis in CRC, thus highlighting the utility of our 3-D culture (3 DC) model for the KRAS-induced development of CRC in 3-D microenvironment. Indeed, using this model, we found that PDE4B is a promising candidate for a therapeutic target as well as prognostic molecular marker in CRC. Further elucidation of the signaling network of PDE4B2 in 3 DC would provide a better understanding of CRC in vivo.

ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response.

ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7Rα and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2Rα expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

KRAS-induced actin-interacting protein regulates inositol 1,4,5-trisphosphate-receptor-mediated calcium release.

KRAS-induced actin-interacting protein (KRAP) was originally characterized as a filamentous- actin-interacting protein. We have recently found that KRAP is an associated molecule with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) and is responsible for the proper subcellular localization of IP(3)R. Since it remains unknown whether KRAP regulates the IP(3)R-mediated Ca(2+) signaling, we herein examined the effects of KRAP on the IP(3)R-mediated Ca(2+) release by Ca(2+) imagings in the cultured HEK293 or MCF7 cells. Reduction of KRAP protein by KRAP-specific siRNA diminishes ATP-induced Ca(2+) release and the ATP-induced Ca(2+) release is completely quenched by the pretreatment with the IP(3)R inhibitor but not with the ryanodine receptor inhibitor, indicating that KRAP regulates IP(3)R-mediated Ca(2+) release. To further reveal mechanistic insights into the regulation of IP(3)R-mediated Ca(2+) release by KRAP, we examined the effects of the KRAP-knockdown on the releasable Ca(2+) content of intracellular Ca(2+) stores. Consequently, reduction of KRAP does not affect the amount of ionophore- or Ca(2+)-ATPase inhibitor-induced Ca(2+) release in the HEK293 cells, indicating that releasable Ca(2+) content of intracellular Ca(2+) stores is not altered by KRAP. Thus, KRAP is involved in the proper regulation of IP(3)R-mediated Ca(2+) release.

Determination of the critical region of KRAS-induced actin-interacting protein for the interaction with inositol 1,4,5-trisphosphate receptor.

KRAS-induced actin-interacting protein (KRAP) was originally characterized as a filamentous-actin-interacting protein. We have recently found that KRAP is an associated molecule with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization and function of IP(3)R. However, the molecular mechanisms underlying the regulation of IP(3)R by KRAP remain elusive. In this report, to determine the critical region of KRAP protein for the regulation of IP(3)R, we generate several mutants of KRAP and examine the association with IP(3)R using coimmunoprecipitation and confocal imaging assays. Coimmunoprecipitations using the deletion mutants reveal that amino-acid residues 1-218 but not 1-199 of KRAP interact with IP(3)R, indicating that the 19-length amino-acid residues (200-218) are essential for the association with IP(3)R. This critical region is highly conserved between human and mouse KRAP. Within the critical region, substitutions of two phenylalanine residues (Phe202/Phe203) in mouse KRAP to alanines result in failure of the association with IP(3)R, suggesting that the two consecutive phenylalanine residues are indispensable for the association. Moreover, the KRAP-knockdown stable HeLa cells exhibit the inappropriate subcellular localization of IP(3)R, in which exogenous expression of full-length of KRAP properly restores the subcellular localization of IP(3)R, but not the 1-218 or 1-236 mutant, indicating that the residual carboxyl-terminal region is also required for the proper subcellular localization of KRAP-IP(3)R complex. All these results provide insight into the understandings for the molecular mechanisms underlying the regulation of IP(3)R, and would reveal a potent strategy for the drug development targeting on IP(3)R.

KRAS-induced actin-interacting protein is required for the proper localization of inositol 1,4,5-trisphosphate receptor in the epithelial cells.

Three inositol 1,4,5-trisphosphate receptor (IP(3)R) subtypes are differentially expressed among tissues and function as the Ca(2+) release channel on specialized endoplasmic reticulum (ER) membranes. The proper subcellular localization of IP(3)R is crucial for its proper function, but this molecular mechanism is unclear. KRAS-induced actin-interacting protein (KRAP) was originally identified as a cancer-related molecule, and is involved in the regulation of whole-body energy homeostasis and pancreatic exocrine system. We herein identified IP(3)R as an associated molecule with KRAP in vivo, and the association was validated by the co-immunoprecipitation and confocal immunostaining studies in mouse tissues including liver and pancreas. The association of KRAP with IP(3)R was also observed in the human epithelial cell lines including HCT116, HeLa and HEK293 cells. Intriguingly, KRAP interacts with distinct subtypes of IP(3)R in a tissue-dependent manner, i.e. IP(3)R1 and IP(3)R2 in the liver and IP(3)R2 and IP(3)R3 in the pancreas. The NH(2)-terminal amino acid residues 1-610 of IP(3)R are critical for the association with KRAP and KRAP-IP(3)R complex resides in a specialized ER but not a typical reticular ER. Furthermore, the localization of particular IP(3)R subtypes in tissues from KRAP-deficient mice is obviously disturbed, i.e. IP(3)R1 and IP(3)R2 in the liver and IP(3)R2 and IP(3)R3 in the pancreas. These findings demonstrate that KRAP physically associates with IP(3)R and regulates the proper localization of IP(3)R in the epithelial cells in vivo and cultured cells, and might shed light on the Ca(2+) signaling underlying physiological cellular programs, cancer development and metabolism-related diseases.

Uterine inversion due to a leiomyoma on postpartum day 41: a case report.

Uterine leiomyomas are common tumors in women of reproductive age and are frequently detected during pregnancy. The major complications during pregnancy include abortion, preterm delivery, abruptio placentae, intrauterine growth retardation, dystocia, and postpartum hemorrhage. Little attention is given to uterine leiomyomas postpartum compared to leiomyomas prior to childbirth. In the present case, a 27-year-old woman, gravida 1 para 1, presented with massive vaginal bleeding, urinary retention and lower abdominal pain on postpartum day 41. She was diagnosed with uterine inversion due to leiomyoma. After a vaginal myomectomy, the uterus was re-placed with a combined vaginal and abdominal approach. Because of timely medical intervention, the patient managed to overcome the crisis and her reproductive organs were successfully preserved.

Zfat-deficiency results in a loss of CD3ζ phosphorylation with dysregulation of ERK and Egr activities leading to impaired positive selection.

The human ZFAT gene was originally identified as a susceptibility gene for autoimmune thyroid disease. Mouse Zfat is a critical transcriptional regulator for primitive hematopoiesis and required for peripheral T cell homeostasis. However, its physiological roles in T cell development remain poorly understood. Here, we generated Zfat (f/f)-LckCre mice and demonstrated that T cell-specific Zfat-deletion in Zfat (f/f)-LckCre mice resulted in a reduction in the number of CD4(+)CD8(+)double-positive (DP) cells, CD4(+)single positive cells and CD8(+)single positive cells. Indeed, in Zfat (f/f)-LckCre DP cells, positive selection was severely impaired. Defects of positive selection in Zfat-deficient thymocytes were not restored in the presence of the exogenous TCR by using TCR-transgenic mice. Furthermore, Zfat-deficient DP cells showed a loss of CD3ζ phosphorylation in response to T cell antigen receptor (TCR)-stimulation concomitant with dysregulation of extracellular signal-related kinase (ERK) and early growth response protein (Egr) activities. These results demonstrate that Zfat is required for proper regulation of the TCR-proximal signalings, and is a crucial molecule for positive selection through ERK and Egr activities, thus suggesting that a full understanding of the precise molecular mechanisms of Zfat will provide deeper insight into T cell development and immune regulation.

Tespa1 is a novel inositol 1,4,5-trisphosphate receptor binding protein in T and B lymphocytes.

Tespa1 has been recently reported to be a critical molecule in T-cell development, however, the precise molecular mechanisms of Tespa1 remain elusive. Here, we demonstrate that Tespa1 shows amino-acid sequence homology to KRAS-induced actin-interacting protein (KRAP), an inositol 1,4,5-trisphosphate receptor (IP3R) binding protein, and that Tespa1 physically associates with IP3R in T and B lymphocytes. Two-consecutive phenylalanine residues (Phe185/Phe186) in Tespa1, which are conserved between Tespa1 and KRAP, are indispensable for the association between Tespa1 and IP3R. These findings suggest that Tespa1 plays critical roles in the immune system through the regulation of the IP3R.

KRAS up-regulates the expression of miR-181a, miR-200c and miR-210 in a three-dimensional-specific manner in DLD-1 colorectal cancer cells.

BACKGROUND: We previously found that oncogenic KRAS induces increased expression of microRNAs (miRNAs), such as miR-200c and miR-221/222, in human colorectal cancer (CRC) HCT116 cells in a three-dimensional (3D)-specific manner, however, the regulation of miRNA expression through oncogenic KRAS in other types of CRC remains unclear. MATERIALS AND METHODS: The differential expression of 94 cancer-related miRNAs was examined in DLD-1 and DKO-4 cells (DLD-1 cells with a disrupted oncogenic KRAS) in 3D cultures. RESULTS: Increased miR-15b, miR-16, miR-23a, miR-24, miR-103 and miR-222 expression was observed in 3D and in 2D cultures. Of note, increased miR-181a, miR-200c and miR-210 expression was only observed in 3D cultures. Furthermore, miR-181a and miR-210 were significantly overexpressed in DLD-1 cells in 3D culture compared with those in HCT116 cells, and were significantly overexpressed in human CRC specimens. CONCLUSION: Oncogenic KRAS regulates 3D-specific miRNAs that are possibly associated with CRC development in vivo.

ALPK2 is crucial for luminal apoptosis and DNA repair-related gene expression in a three-dimensional colonic-crypt model.

BACKGROUND: Oncogenic KRAS signaling is dysregulated in a three-dimensional (3D)-specific manner in human colorectal cancer (CRC) HCT116 cells. However, the identity of the crucial genes which are down-regulated through oncogenic KRAS in 3D cultures remains unclear. MATERIALS AND METHODS: We established a specific anti-alpha-kinase 2 (ALPK2) antibody and addressed the ALPK2 function in HKe3 cells, which are HCT116 cells with a disruption in oncogenic KRAS, in a 3D colonic-crypt model. RESULTS: In HKe3 cells grown in 3D culture, ALPK2 siRNA inhibited luminal apoptosis and reduced the expression of cleaved caspase-3. Furthermore, ALPK2 siRNA reduced the expression of DNA repair genes. Reduced expression of ALPK2 mRNA was found to be correlated with clinical colorectal adenomas in a public dataset of gene expression analyses. CONCLUSION: ALPK2, down-regulated by oncogenic KRAS, is crucial for luminal apoptosis and expression of DNA repair-related genes, possibly in the transition of normal colonic crypt to adenoma.

Oncogenic KRAS regulates miR-200c and miR-221/222 in a 3D-specific manner in colorectal cancer cells.

BACKGROUND: Oncogenic KRAS plays several key roles in a three-dimensional (3D) colonic-crypt model. However, miRNA expression regulated by oncogenic KRAS in this model is still elusive. MATERIALS AND METHODS: The differential expression of 105 cancer-related microRNAs was examined and compared in HCT116 cells and HKe3 cells (HCT116 cells in which mutated KRAS allele was deleted) in 3D culture. HKe3 cells stably overexpressing oncogenic KRAS and the public datasets for microRNA expression analysis of colorectal cancer were further examined. RESULTS: The increased expression of miR-200c, miR-221 and miR-222 were observed exclusively in 3D culture, but not in the two-dimensional culture. These microRNAs were regulated by oncogenic KRAS and were significantly overexpressed in human colorectal tumor specimens. Of note, the protein expression level of Phosphatase and tensin homolog (PTEN), a putative target of miR-221/222 cluster, was reduced under the control of oncogenic KRAS in a 3D-specific manner. CONCLUSION: Oncogenic KRAS regulates 3D-specific molecules, possibly being associated with colorectal tumor development in vivo.

KRAS-mediated up-regulation of RRM2 expression is essential for the proliferation of colorectal cancer cell lines.

BACKGROUND: We previously investigated the mRNA expression of colorectal cancer cell lines via a microarray analysis and found several genes that were significantly up-regulated by oncogenic KRAS under serum-starved conditions. Of these genes, we focused on ribonucleotide reductase M2 (RRM2), which was reported to be associated with DNA synthesis. MATERIALS AND METHODS: Cell proliferation and colony formation assays were performed using HCT116 cells transfected with lentiviral RRM2-shRNAs. RESULTS: Under serum-starved conditions, the expression level of RRM2 protein increased in HCT116 cells compared to HKe3 cells (HCT116 cells with a disruption in oncogenic KRAS), and the re-expression of KRAS in HKe3 cells induced the expression of RRM2. Both the cell proliferation under serum-depleted conditions and the anchorage-independent growth were impaired by the reduction of RRM2 protein expression. CONCLUSION: RRM2 represents a novel therapeutic target, thus highlighting the potential utility of RRM2 inhibitors in colorectal cancer with oncogenic KRAS.