Ming-Ji Fann

Regulation of IGFBP-5 expression during tumourigenesis and differentiation of oral keratinocytes

To identify molecular events involved in the pathogenesis of oral squamous cell carcinoma (OSCC), genes differentially expressed in OSCC and non‐cancerous matched tissue (NCMT) samples were analysed using a subtractive hybridization strategy. NCMT‐enriching clones that have been linked to suppressor pathway in previous studies were subjected to advanced analyses. Complete absence of insulin‐like growth factor binding protein‐5 (IGFBP‐5) expression at both the mRNA and the protein level was identified in nearly all (5/6) OSCC cell lines with the exception of the SCC25 cell line, which exhibited high IGFBP‐5 expression. However, this protein is consistently present in cultured normal human oral keratinocytes (NHOKs). Immunohistochemistry revealed moderate to strong cytoplasmic immunoreactivity of IGFBP‐5 in the stratum spinosum and stratum granulosum in the vast majority of NCMT samples. A remarkable reduction in IGFBP‐5 immunoreactivity was detected in 56% (26/46) of OSCC samples, compared with the corresponding NCMT (p < 0.0001). Induction of differentiation in both NHOKs and SCC25 up‐regulated IGFBP‐5 expression. Administration of a green tea compound with anti‐cancer properties, (–)‐epigallocatechin 3‐gallate, at a concentration of 5–20 µg/ml also up‐regulated IGFBP‐5 expression in NHOKs in a dose‐dependent manner. The findings suggest that IGFBP‐5 may be an important factor in the differentiation of oral keratinocytes and that down‐regulation of IGFBP‐5 may be involved in the neoplastic transformation of oral keratinocytes. Copyright

The inhibitor ABIN-2 disrupts the interaction of receptor-interacting protein with the kinase subunit IKKgamma to block activation of the transcription factor NF-kappaB and potentiate apoptosis.

NF-kappaB (nuclear factor kappaB) proteins are key transcription factors that regulate gene expression in response to various extracellular stimuli. The pathway leading to the activation of NF-kappaB involves a complicated network that includes a number of signalling molecules. The recent identification of a wide range of negative regulators of NF-kappaB has given another layer of complexity in NF-kappaB activation. We and others have previously identified the protein ABIN-2 (A20 binding inhibitor of NF-kappaB 2) as an inhibitor of NF-kappaB activation. In the present paper, we demonstrate that ABIN-2 exerts its inhibitory function by blocking the interaction of RIP (receptor-interacting protein) with the downstream effector IKKgamma, a non-kinase component of the IkappaB (inhibitory kappaB) kinase complex. When overexpressed in cells, ABIN-2 bound to IKKgamma and prevented the association of IKKgamma with RIP. By a deletion mapping, a stretch of 50 amino acids on ABIN-2 is found to be essential for its interaction with IKKgamma. The ABIN-2 mutant that lacked these 50 amino acids did not interact with IKKgamma and, consequently, failed to inhibit NF-kappaB activation. Strikingly, a portion of RIP, which is similar to this 50-residue domain of ABIN-2, is also essential for RIP interaction with IKKgamma. The RIP mutant with deletion of this similar region did not associate with IKKgamma and had substantial reduction of its ability to mediate NF-kappaB activation. Taken together, these conserved 50 residues of ABIN-2 and RIP define a novel structural domain in mediating a key step in the NF-kappaB signalling pathway through the interaction with IKKgamma. Finally, the signalling pathway of NF-kappaB activation is known to promote survival in many cellular events. The mechanism for decision between cell death and survival is under fine regulation. In the present paper, we demonstrated further that the expression of ABIN-2 could promote the RIP-mediated apoptosis by presumably suppressing the anti-apoptotic effect of NF-kappaB.

Regulation of sodium-calcium exchanger activity by creatine kinase under energy-compromised conditions.

Na+/Ca2+ exchanger (NCX) is one of the major mechanisms for removing Ca2+ from the cytosol especially in cardiac myocytes and neurons, where their physiological activities are triggered by an influx of Ca2+. NCX contains a large intracellular loop (NCXIL) that is responsible for regulating NCX activity. Recent evidence has shown that proteins, including kinases and phosphatases, associate with NCX1IL to form a NCX1 macromolecular complex. To search for the molecules that interact with NCX1IL and regulate NCX1 activity, we used the yeast two-hybrid method to screen a human heart cDNA library and found that the C-terminal region of sarcomeric mitochondrial creatine kinase (sMiCK) interacted with NCX1IL. Moreover, both sMiCK and the muscle-type creatine kinase (CKM) coimmunoprecipitated with NCX1 using lysates of cardiacmyocytes and HEK293T cells that transiently expressed NCX1 and various creatine kinases. Both sMiCK and CKM were able to produce a recovery in the decreased NCX1 activity that was lost under energy-compromised conditions. This regulation is mediated through a putative PKC phosphorylation site of sMiCK and CKM. The autophosphorylation and the catalytic activity of sMiCK and CKM are not required for their regulation of NCX1 activity. Our results suggest a novel mechanism for the regulation of NCX1 activity.Na(+)/Ca(2+) exchanger (NCX) is one of the major mechanisms for removing Ca(2+) from the cytosol especially in cardiac myocytes and neurons, where their physiological activities are triggered by an influx of Ca(2+). NCX contains a large intracellular loop (NCXIL) that is responsible for regulating NCX activity. Recent evidence has shown that proteins, including kinases and phosphatases, associate with NCX1IL to form a NCX1 macromolecular complex. To search for the molecules that interact with NCX1IL and regulate NCX1 activity, we used the yeast two-hybrid method to screen a human heart cDNA library and found that the C-terminal region of sarcomeric mitochondrial creatine kinase (sMiCK) interacted with NCX1IL. Moreover, both sMiCK and the muscle-type creatine kinase (CKM) coimmunoprecipitated with NCX1 using lysates of cardiacmyocytes and HEK293T cells that transiently expressed NCX1 and various creatine kinases. Both sMiCK and CKM were able to produce a recovery in the decreased NCX1 activity that was lost under energy-compromised conditions. This regulation is mediated through a putative PKC phosphorylation site of sMiCK and CKM. The autophosphorylation and the catalytic activity of sMiCK and CKM are not required for their regulation of NCX1 activity. Our results suggest a novel mechanism for the regulation of NCX1 activity.

Characterization of Ca2+ signaling pathways in mouse adrenal medullary chromaffin cells.

J. Neurochem. (2010) 112, 1210–1222.

Cdk12 and Cdk13 regulate axonal elongation through a common signaling pathway that modulates Cdk5 expression.

Cdk12 and Cdk13 are Cdc2-related proteins that share 92% identity in their kinase domains. Using in situ hybridization and Western blot analysis, we detected the expression of Cdk12 and Cdk13 mRNAs and their proteins in developing mouse embryos, especially during development of the nervous system. We explored the roles of Cdk12 and Cdk13 in neuronal differentiation using the P19 neuronal differentiation model. Upon knockdown of Cdk12 or Cdk13, no effect on differentiated cell numbers was detected, but a substantial decrease of numbers of neurons with long neurites was identified. Similarly, knockdown of Cdk12 or Cdk13 in primarily cultured cortical neurons shortens the averaged axonal length. A microarray analysis was used to examine changes in gene expression after knockdown or overexpression of Cdk12 and we identified Cdk5 as a molecule potentially involved in mediating the effect of Cdk12 and Cdk13. Depletion of Cdk12 or Cdk13 in P19 cells significantly reduces Cdk5 expression at both the mRNA and protein levels. Expression of Cdk5 protein in the developing mouse brain is also reduced in conditional Cdk12-knockout mice in proportion to the residual amount of Cdk12 protein present. This suggests that the reduced axonal outgrowth after knockdown of Cdk12 or Cdk13 might be due to lower Cdk5 expression. Furthermore, overexpression of Cdk5 protein in P19 cells was able to partially rescue the neurite outgrowth defect observed when Cdk12 or Cdk13 is depleted. Together, these findings suggest that Cdk12 and Cdk13 regulate axonal elongation through a common signaling pathway that modulates Cdk5 expression.

Change of gene expression profiles in the retina following optic nerve injury

The purpose of the present study was to search for changes in gene expression patterns in the retina following optic nerve injury. We conducted a subtractive hybridization for comparison of the mRNAs in those retinas receiving optic nerve crush injury and those receiving sham operation. Both forward and reverse subtractions were carried out for 8-h and 24-h time points postoperatively. Resultant subtractive cDNA for each group was re-amplified and cloned to a plasmid. After DNA sequencing, the identity of subtractive cDNA was analyzed by blasting sequences to the Nonredundant gene database, Unigene database, and dbest database at NCBI. Thirty-four known genes and 32 EST were found in the forward subtractions. Forty-two known genes and 46 EST were found in reverse subtractions. Identities of the rest could not been found in the databases. To verify the subtractive results, RT-PCR was performed to test expression patterns of eight known genes found in the above analysis. Among these eight genes, seven demonstrated a statistically significant difference between the crushed eyes and the control eyes by quantitative image analysis. Together, our data show that expression of fatso, ephrin B2, NonO, Zfx, vitronectin, and XLRS increased after optic nerve injury, and expression of stathmin exhibited reduction after injury.

Novel Mutations of the OPA1 Gene in Chinese Dominant Optic Atrophy

PURPOSE To investigate OPA1 gene mutations in Chinese patients with autosomal dominant optic atrophy and sporadic optic atrophy. DESIGN Molecular genetic studies and observational case series. PARTICIPANTS Twenty-four patients from 10 unrelated Chinese pedigrees of autosomal-dominant optic atrophy, 35 isolated cases with bilateral optic atrophy of unknown cause, and 50 unrelated normal controls. METHODS Genomic DNA was extracted from peripheral blood leukocytes. All 28 coding exons of the OPA1 gene and flanking intron splice sites were sequenced. Putative mutations were reexamined for segregation in the respective families by direct sequencing. Further characterization of selected splicing site mutations was performed by reverse transcription-polymerase chain reaction (PCR) of each patients leukocyte mRNA. MAIN OUTCOME MEASURES Direct sequencing of the OPA1 gene. RESULTS Four OPA1 gene mutations were detected, including 2 splicing site mutations (c.1065+2T>C on intron 10 and c.1212+2insT on intron 12), 1 deletion (c.1776_1778delACT on exon 19), and 1 missense mutation (c.2846 T>C on exon 28). The c.1212+2insT, c.1776_1778delACT, and c.2846T>C mutations were newly identified OPA1 mutations. Reverse transcription (RT)-PCR and direct sequencing revealed that the splicing site mutations on c.1065+2T>C and c.1212+2insT caused skipping of exons 10 and 12, respectively. The c.1776_1778delACT mutation led to a deletion of the Leu amino acid on residue 593. OPA1 mutations were found in 4 of 10 familial cases (40 %) and in 1 of 35 sporadic cases of optic atrophy. CONCLUSIONS OPA1 gene mutations are causative in Chinese autosomal-dominant optic atrophy and sporadic optic atrophy. Screening for OPA1 gene mutations in patients with childhood onset optic atrophy who have no affected relatives is useful in making the diagnosis.

DIFFERENTIAL EXPRESSION OF PROTEIN KINASES IN CULTURED PRIMARY NEURONS DERIVED FROM THE CEREBRAL CORTEX, HIPPOCAMPUS, AND SYMPATHETIC GANGLIA

Protein kinases play pivotal roles in the development of the nervous system. They are involved in almost every stage of neuronal development, from initial proliferation and differentiation of progenitor cells to pathfinding of neurites and formation of synapses. Activation of protein kinases is also critical for neuronal cell survival. To gain further insights into kinases in neural development, we studied the expression patterns of protein kinases in three cultured primary neurons by degenerate primer-based reverse transcription-polymerase chain reaction (PCR) and DNA sequencing, taking advantage of all known kinases containing a conserved catalytic domain. Our data demonstrated that the expression patterns of kinases in various cultured neurons are not only different from those of non-neural tissues, but also distinct among neurons derived from discrete origins. For example, FGF receptor 1 is predominantly expressed in hippocampal neurons. As this approach may be biased during PCR and cloning steps, an RNase protection assay was employed to verify the expression levels of six kinases in cultured neurons. Results from the RNase protection assay did generally confirm those obtained by the PCR-based method. However, quantitative nature of the latter was dependent on numbers of clones analyzed, and discrepancy of expression levels of kinases detected by the two methods was sometimes observed.

ENU mutagenesis identifies mice modeling Warburg Micro Syndrome with sensory axon degeneration caused by a deletion in Rab18.

Mutations in the gene of RAB18, a member of Ras superfamily of small G-proteins, cause Warburg Micro Syndrome (WARBM) which is characterized by defective neurodevelopmental and ophthalmological phenotypes. Despite loss of Rab18 had been reported to induce disruption of the endoplasmic reticulum structure and neuronal cytoskeleton organization, parts of the pathogenic mechanism caused by RAB18 mutation remain unclear. From the N-ethyl-N-nitrosourea (ENU)-induced mutagenesis library, we identified a mouse line whose Rab18 was knocked out. This Rab18(-/-) mouse exhibited stomping gait, smaller testis and eyes, mimicking several features of WARBM. Rab18(-/-) mice were obviously less sensitive to pain and touch than WT mice. Histological examinations on Rab18(-/-) mice revealed progressive axonal degeneration in the optic nerves, dorsal column of the spinal cord and sensory roots of the spinal nerves while the motor roots were spared. All the behavioral and pathological changes that resulted from abnormalities in the sensory axons were prevented by introducing an extra copy of Rab18 transgene in Rab18(-/-) mice. Our results reveal that sensory axonal degeneration is the primary cause of stomping gait and progressive weakness of the hind limbs in Rab18(-/-) mice, and optic nerve degeneration should be the major pathology of progressive optic atrophy in children with WARBM. Our results indicate that the sensory nervous system is more vulnerable to Rab18 deficiency and WARBM is not only a neurodevelopmental but also neurodegenerative disease.

Cell Death Caused by the Synergistic Effects of Zinc and Dopamine is Mediated by a Stress Sensor Gene Gadd45b ‐ Implication in the Pathogenesis of Parkinson's Disease

The pathogenesis of Parkinsons disease (PD) is not completely understood, Zinc (Zn2+) and dopamine (DA) have been shown to involve in the degeneration of dopaminergic cells. By microarray analysis, we identified Gadd45b as a candidate molecule that mediates Zn2+ and DA‐induced cell death; the mRNA and protein levels of Gadd45b are increased by Zn2+ treatment and raised to an even higher level by Zn2+ plus DA treatment. Zn2+ plus DA treatment‐induced PC12 cell death was enhanced when there was over‐expression of Gadd45b and was decreased by knock down of Gadd45b. MAPK p38 and JNK signaling was able to cross‐talk with Gadd45b during Zn2+ and DA treatment. The synergistic effects of Zn2+ and DA on PC12 cell death can be accounted for by an activation of the Gadd45b‐induced cell death pathway and an inhibition of p38/JNK survival pathway. Furthermore, the in vivo results show that the levels of Gadd45b protein expression and phosphorylation of p38 were increased in the substantia nigra by the infusion of Zn2+/DA in the mouse brain and the level of Gadd45b mRNA is significantly higher in the substantia nigra of male PD patients than normal controls. The novel role of Gadd45b and its interactions with JNK and p38 will help our understanding of the pathogenesis of PD and help the development of future treatments for PD.