5'-UTR SNP of FGF13 causes translational defect and intellectual disability.

The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5'-untranslated region (5'-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5'-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5'-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5'-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.

Activin C expressed in nociceptive afferent neurons is required for suppressing inflammatory pain.

Emerging evidence suggests that the suppressive modulators released from nociceptive afferent neurons contribute to pain regulation. However, the suppressive modulators expressed in small-diameter neurons of the dorsal root ganglion remain to be further identified. The present study shows that the activin C expressed in small dorsal root ganglion neurons is required for suppressing inflammation-induced nociceptive responses. The expression of activin C in small dorsal root ganglion neurons of rats was markedly downregulated during the early days of peripheral inflammation induced by intraplantar injection of the complete Freund's adjuvant. Intrathecal treatment with the small interfering RNA targeting activin ßC or the antibodies against activin C could enhance the formalin-induced nociceptive responses, and impair the recovery from the complete Freund's adjuvant-induced thermal hyperalgesia. Intrathecally applied activin C could reduce nociceptive responses induced by formalin or complete Freund's adjuvant. Moreover, activin C was found to inhibit the inflammation-induced phosphorylation of extracellular signal-regulated kinase in the dorsal root ganglia and the dorsal spinal cord. Thus, activin C functions as an endogenous suppressor of inflammatory nociceptive transmission and may have a therapeutic potential for treatment of inflammatory pain.

Follistatin-like 1 suppresses sensory afferent transmission by activating Na+,K+-ATPase.

Excitatory synaptic transmission is modulated by inhibitory neurotransmitters and neuromodulators. We found that the synaptic transmission of somatic sensory afferents can be rapidly regulated by a presynaptically secreted protein, follistatin-like 1 (FSTL1), which serves as a direct activator of Na(+),K(+)-ATPase (NKA). The FSTL1 protein is highly expressed in small-diameter neurons of the dorsal root ganglion (DRG). It is transported to axon terminals via small translucent vesicles and secreted in both spontaneous and depolarization-induced manners. Biochemical assays showed that FSTL1 binds to the α1 subunit of NKA and elevates NKA activity. Extracellular FSTL1 induced membrane hyperpolarization in cultured cells and inhibited afferent synaptic transmission in spinal cord slices by activating NKA. Genetic deletion of FSTL1 in small DRG neurons of mice resulted in enhanced afferent synaptic transmission and sensory hypersensitivity, which could be reduced by intrathecally applied FSTL1 protein. Thus, FSTL1-dependent activation of NKA regulates the threshold of somatic sensation.

Potentiation of excitatory transmission in substantia gelatinosa neurons of rat spinal cord by inhibition of estrogen receptor alpha.

BACKGROUND: It has been shown that estrogen is synthesized in the spinal dorsal horn and plays a role in modulating pain transmission. One of the estrogen receptor (ER) subtypes, estrogen receptor alpha (ERα), is expressed in the spinal laminae I-V, including substantia gelatinosa (SG, lamina II). However, it is unclear how ERs are involved in the modulation of nociceptive transmission. RESULTS: In the present study, a selective ERα antagonist, methyl-piperidino-pyrazole (MPP), was used to test the potential functional roles of spinal ERα in the nociceptive transmission. Using the whole-cell patch-clamp technique, we examined the effects of MPP on SG neurons in the dorsal root-attached spinal cord slice prepared from adult rats. We found that MPP increased glutamatergic excitatory postsynaptic currents (EPSCs) evoked by the stimulation of either Aδ- or C-afferent fibers. Further studies showed that MPP treatment dose-dependently increased spontaneous EPSCs frequency in SG neurons, while not affecting the amplitude. In addition, the PKC was involved in the MPP-induced enhancement of synaptic transmission. CONCLUSIONS: These results suggest that the selective ERα antagonist MPP pre-synaptically facilitates the excitatory synaptic transmission to SG neurons. The nociceptive transmission evoked by Aδ- and C-fiber stimulation could be potentiated by blocking ERα in the spinal neurons. Thus, the spinal estrogen may negatively regulate the nociceptive transmission through the activation of ERα.

Coexpression of delta- and mu-opioid receptors in nociceptive sensory neurons.

Morphine-induced analgesia and antinociceptive tolerance are known to be modulated by interaction between delta-opioid receptors (DORs) and mu-opioid receptors (MORs) in the pain pathway. However, evidence for expression of DORs in nociceptive small-diameter neurons in dorsal root ganglia (DRG) and for coexistence of DORs with MORs and neuropeptides has recently been challenged. We now report, using in situ hybridization, single-cell PCR, and immunostaining, that DORs are widely expressed not only in large DRG neurons but in small ones and coexist with MORs in peptidergic small DRG neurons, with protachykinin-dependent localization in large dense-core vesicles. Importantly, both DOR and MOR agonists reduce depolarization-induced Ca(2+) currents in single small DRG neurons and inhibit afferent C-fiber synaptic transmission in the dorsal spinal cord. Thus, coexistence of DORs and MORs in small DRG neurons is a basis for direct interaction of opioid receptors in modulation of nociceptive afferent transmission and opioid analgesia.

[Effect of leptin on growth hormone secretion and apoptosis of GH3 cells].

In order to investigate the effect of leptin on the secretion of rat pituitary adenoma GH3 cell and its mechanisms, we observed the effect of leptin on the growth hormone secretion, proliferation and apoptosis of GH3 cells. The results indicated that leptin at 1, 10, and 100 nmol/L could inhibit the basal growth hormone secretion of GH3 cells in a dose dependent manner (P<0.05). Short-term treatment of leptin (10 nmol/L) for 30 min, 1 and 3 h did not affect basal GH secretion. However, treatment of the GH3 cells with leptin (10 nmol/L) for 1 d or longer resulted in an inhibition of GH secretion (P<0.05). We used MTT method and flow cytometery (FCM) to study the effect of leptin on the proliferation and apoptosis of GH3 cells. We found that leptin inhibited proliferation of GH3 cells with a dose-dependent manner. And leptin reduced the proportion of cells in S phase, increased the proportion of cells in G1, and increased the proportion of GH3 cells in 2 and 4 phase. These results demonstrate that leptin inhibits the basal GH secretion of GH3 cells, which may be due to the inhibition of DNA synthesis and advanced apoptosis of GH3 cells.

Estrogen influences the differentiation, maturation and function of dendritic cells in rats with experimental autoimmune encephalomyelitis.

AIM: To examine if estrogen can affect the immune response at the dendritic cells (DCs) level in rats with experimental autoimmune encephalomyelitis (EAE). METHODS: Lewis rats were immunized with inoculum containing MBP(68-86). DCs were derived from spleen monocytes of EAE rats with IL-4 and GM-CSF in presence of 17 beta-estradiol (E2). Nitric oxide (NO) was detected by Griess reagent. The surface markers and cytokines production of DCs were shown by flow cytometry. DCs were cocultured with MBP-specific T cells, [(3)H]-TdR incoportation was used to reveal the antigen presentability, the supernatant of the coculture were collected to examine the cytokines secretion by ELISA. RESULTS: E2 activated DCs by accelerating the maturation process characterized by upregulation of MHC II and costimulating molecule B7-1, B7-2, drastic high expression of CD40. IFN-kappa-producing DCs were also elevated without any alteration of IL-10. Estradiol-treated DCs (E2-DCs) secreted more NO in the culture supernatant. By contrast, E2-DCs showed decreased antigen presentation ability with reduced secretion of IFN-kappa but no alteration of IL-10 in the coculture with T cells. CONCLUSION: Estrogen can affect the differentiation, maturation and function of DCs from EAE rats, which may be attributed to its protection against EAE and the remission of multiple sclerosis patients in pregnancy.

[Enhancement effect of IL-2 on the proliferation of cultured mammary carcinoma cell line Bcap-37].

AIM: To investigate the effects of IL-2 on the proliferation of human mammary carcinoma cell line Bcap-37 and explore its possible mechanism. METHODS: Enhancement effect of IL-2 on proliferation of cultured Bcap-37 cells, the IL-2 expression on the cells, the expressions of IL-2Ralpha, beta, gamma mRNAs in the cells, the effect of IL-2 on DNA content at various periods of cell cycle and on Ca(2+) concentration in the cells were detected or observed by MTT colorimetry, immunohistochemical staining, RT-PCR, flow cytometry and laser scanning confocal microscope, respectively. RESULTS: IL-2 of 1x10(5) to 1x10(6) U/L significantly stimulated the proliferation of cultured Bcap-37 cells. IL-2 was secreted and IL-2Ralpha, beta and gamma were expressed by cultured Bcap-37 cells. IL-2(1x10(5) U/L) increased ratio of Bcap-37 cells in G(2) and S phases and decreased the Ca(2+) release from Bcap-37 cells. CONCLUSION: IL-2 significantly enhance the proliferation of mammary carcinoma cell line Bcap-37. The enhancement effect of IL-2 on Bcap-37 cell proliferation is possibly related to the expression of IL-2R and decreased Ca(2+) concentration in the cells.

[Effect of tamoxifen on proliferation of cultured breast cancer and cervical carcinoma cell lines].

AIM: To investigate the effects of tamoxifen on proliferation of human breast cancer Bcap-37 cells and cervical carcinoma HeLa cells and to explore it's possible mechanism. METHODS: The techniques of cell culture, growth curves, flow cytometry and laser scanning confocal microscope were used. RESULTS: Tamoxifen (10(-6) mol/L) shifted the growth curve of Bcap-37 cells downward, and shifted the growth curve of HeLa cells upward. Tamoxifen (10(-8) - 10(-6) mol/L) inhibited the proliferation of Bcap-37 cells in a dose-dependent manner, but stimulated the proliferation of HeLa cells in a dose-dependent manner. Bcap-37 cells appeared apoptosis when treated with tamoxifen (10(-6) mol/L), and the same dose stimulated the proliferation of HeLa cells at GI/S phases. The apoptotic rate of Bcap-37 cells was 97.5%. It blocked G1 phase of HeLa cells from 55.5% to 32.8%, and increased the S phase from 29.0% to 49.4%. Tamoxifen (10(-6) mol/L) also increased the releasing of calcium in Bcap-37 and HeLa cells. CONCLUSION: Tamoxifen can significantly influence the proliferation of breast cancer and cervical carcinoma cells possibly by affecting cell cycle and stimulating the releasing of Ca2+ in the cells.

[Expression of estrogen receptors alpha and beta in human tongue squamous cancer cell and influence of beta-estradiol on the proliferation of tongue cancer cell].

AIM: To observe the expression of estrogen receptors alpha and beta in human tongue squamous cancer line Tca8113 cell, and to study the influence of beta-estradiol (beta-E2) on the proliferation and cell cycle of cultured Tca8113 cell. METHODS: Immunocytochemistry and RT-PCR methods were used to observe the expression of estrogen receptors (ER) in human tongue squamous carcinoma line Tca8113 cell. 3H-TdR incorporation and cell cycle analysis were used to examine the change of proliferation and DNA synthesis of Tca8113 cell. RESULTS: ER-alpha and ER-beta mRNA were expressed in human tongue squamous cancer cell, and the expression of ER-beta was weaker than that of ER-alpha. beta-Estradiol at 10(-8) mol/L - 10(-6) mol/L could increase the proliferation of human tongue squamous carcinoma cell in a dose dependent manner (P < 0.01). beta-E2 (10(-6) mol/L) could increase the proportion of cells in S phase and G2 phase from 23.5% up to 37.7%. The effect of estradiol on the proliferation of cultured human tongue squamous cancer line Tca8113 cell could be inhibited by Tamoxifen. CONCLUSION: There are ER-alpha and ER-beta expression in human tongue squamous cancer line Tca8113 cell, and beta-estradiol promotes the proliferation and cell cycle of cultured human Tca8113 cell.

[Expression of interleukin-2 receptors on breast cancer cell line MCF-7 cells].

AIM: To investigate the expression of interleukin-2 receptors (IL-2Rs) on MCF-7 cells, estradiol's regulation of IL-2Rs expression and the influence of IL-2 on the proliferation of MCF-7 cells. METHODS: Immunocytochemistry and flow cytometric analysis were used to investigate the expression of interleukin-2 receptors (IL-2Rs) by using of specific IL-2R polyclonal antibody; MTT method and 3H-TdR incorporation method were used to examine the changes of proliferation of MCF-7 cells. RESULTS: IL-2Ralpha, beta, gamma like immunoreactive substances can be found on MCF-7 cells and the IL-2Rgamma immunostaining was more strong than the other two. Estradiol of 10(-6) mol/L can increase the percentage of immunoreactive cells of IL-2Ralpha, beta and the expression of IL-2Rgamma. Exogenous addition of recombinant IL-2 of 100 U/ml to 1 000 U/ml can significantly increase the proliferation of MCF-7 cells. CONCLUSION: MCF-7 cell can express IL-2R and estradiol can regulate their expression, IL-2 can influence the proliferation of MCF-7 cells.