All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38␣/␤/␥ MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38␥ MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38␥ MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.

Transamidation is a post-translational modification of proteins mediated by tissue transglutaminase II (TGase), a GTP-binding protein, participating in signal transduction pathways as a non-conventional G-protein. Retinoic acid (RA), which is known to have a role in cell differentiation, is a potent activator of TGASE: The activation of TGase results in increased transamidation of RhoA, which is inhibited by monodansylcadaverine (MDC; an inhibitor of transglutaminase activity) and TGaseM (a TGase mutant lacking transglutaminase activity). Transamidated RhoA functions as a constitutively active G-protein, showing increased binding to its downstream target, RhoA-associated kinase-2 (ROCK-2). Upon binding to RhoA, ROCK-2 becomes autophosphorylated and demonstrates stimulated kinase activity. The RA-stimulated interaction between RhoA and ROCK-2 is blocked by MDC and TGaseM, indicating a role for transglutaminase activity in the interaction. Biochemical effects of TGase activation, coupled with the formation of stress fibers and focal adhesion complexes, are proposed to have a significant role in cell differentiation.

Rho GTPases such as RhoA, Rac1 and Cdc42 are crucial players in the regulation of signal transduction pathways required for neuronal differentiation. Using an in vitro cell culture model of neuroblastoma SH-SY5Y cells, we demonstrated previously that RhoA is an in vivo substrate of tissue transglutaminase (TGase) and retinoic acid (RA) promoted activation of RhoA by transamidation. Although activation of RhoA promoted cytoskeletal rearrangement in SH-SY5Y cells, it was not involved in induction of neurite outgrowth. Here, we demonstrate that RA promotes activation of Rac1 in SH-SY5Y cells in a transamidation-independent manner. RA-induced activation of Rac1 is mediated by phosphatidylinositol 3-kinase (PI3K), probably because of phosphorylation of the p85 regulatory subunit by Src kinases. Over-expression of constitutively active PI3K or Rac1-V12 induces neurite outgrowth, activation of mitogen activated protein kinases (MAPKs), and expression of neuronal markers. The PI3K inhibitor LY294002, or over-expression of dominant negative Rac1-N17, blocks RA-induced neurite outgrowth, activation of MAPKs, and expression of neuronal markers, suggesting that activation of PI3K/Rac1 signaling represents a potential mechanism for regulation of neuronal differentiation in SH-SY5Y cells.

Renal transplantation (RTx) recipients have a high incidence of cancer, including transitional cell carcinoma (TCC). Posttransplantation urologic malignancies still present a challenge for transplant surgeons. Using the Dialysis and Transplant Registry of Taichung Veterans General Hospital, a total of 55 cancers were diagnosed in 52 RTx recipients between May 1983 and September 2001. Of these, 24 RTx recipients developing TCC were identified and presented the distinctly high percentage (43.6%) of TCC that were malignancies after RTx in Taiwan. The mean time between transplantation and initial diagnosis was 46 months in our series. Painless hematuria with pyuria is the most common mode of presentation. Transitional cell carcinoma of RTx recipients had multiple foci. Moreover, synchronous TCC in bilateral upper urinary tracts were confirmed in 9 (41%) recipients. The pathologic status of disease is invasive at diagnosis (pTa: 2, pT1: 7, pT2: 4, pT3: 6, pT4: 2, graft metastasis: 1 and distant metastasis: 2). Disseminated metastasis occurred in 6 recipients, all of whom died of their disease within 16 months. Five recipients received adjuvant chemotherapy and retained stable renal function. We conclude that RTx recipients have a markedly increased incidence of TCC in Taiwan, and that prophylactic bilateral nephroureterectomy of native kidneys with bladder cuff excision can be performed simultaneously in RTx recipients with TCC.

Intra-abdominal retained surgical sponge is an uncommon surgical error. Herein, we report a 92-year-old woman who was brought to the emergency room for acute urinary retention. She had a history of vaginal hysterectomy for uterine prolapse 18 years previously, performed at our hospital. Retained surgical sponge in the pelvic cavity was suspected by abdominal computed tomography. The surgical gauze was removed by laparotomy excision and the final diagnosis was gossypiboma.