Post-transcriptional regulation of CLMP mRNA is controlled by tristetraprolin in response to TNFalpha via c-Jun N-terminal kinase signalling.

During spermatogenesis, extensive restructuring of blood-testis barrier takes place to facilitate the migration of preleptotene/leptotene spermatocytes from the basal to the adluminal compartment in the seminiferous epithelium. However, the biochemical mechanisms involved in this event remain elusive. Recent studies have shown that pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) plays a crucial role in this event by inhibiting the expression of tight junction proteins in Sertoli cells. In the present study, we sought to examine the detailed mechanism on how TNFalpha affects the expression of CLMP (coxsackie- and adenovirus-receptor-like membrane protein), a newly identified tight junction transmembrane protein, in the testis. Addition of TNFalpha (10 ng/ml) to Sertoli cell culture (TM4 cells) significantly reduced the steady-state CLMP mRNA and protein levels. In an mRNA stability assay, it was shown that the rate of CLMP mRNA degradation was significantly increased when cells treated with TNFalpha were compared with vehicle. Blockage of the JNK (c-Jun N-terminal kinase) signalling pathway by SP600125 significantly abolished the TNFalpha-mediated destabilization of CLMP mRNA. Activation of the JNK signalling pathway by TNFalpha up-regulated the expression of an RNA-binding protein, TTP (tristetraprolin). TTP was present in the RNA-protein complex in the RNA EMSA (electrophoretic mobility shift assay) and decreased the CLMP 3'-UTR (untranslated region)-dependent luciferase activity. Taken together, these results suggest that the TNFalpha-mediated mRNA degradation of the CLMP gene is controlled by TTP through the JNK signalling cascade.

Expression of CLMP, a novel tight junction protein, is mediated via the interaction of GATA with the Kruppel family proteins, KLF4 and Sp1, in mouse TM4 Sertoli cells.

Regulation of tight junction protein expressions in Sertoli cells is important for germ cell translocation across the blood-testis-barrier (BTB) during spermatogenesis. In this study, a novel tight junction transmembrane protein, CLMP, found expressed in mouse testis was shown to localize at the BTB along with the tight junction marker ZO-1. By the use of transient transfection assay performed in a mouse Sertoli cell-cell line, TM4 cells, we showed that the minimal CLMP promoter was located between nucleotides -550 and -288 relative to the translation start site. Site-directed mutagenic studies showed that three motifs, namely GATA, KLF4, and SRY, within this region functionally co-operated with one another to regulate CLMP gene transcription. Using specific antibodies in EMSA analysis, a ternary protein complex GATA-1/GATA-6/KLF4 was detected at all the three motifs, suggesting that a looping mechanism might involve in regulating CLMP gene transcription. Interestingly, the ubiquitously expressed transcription factors, Sp1 and Sp3, were also found in this ternary complex over the KLF4 motif. Overexpression of KLF4 significantly increased the promoter activity whilst overexpression of Sp1 or Sp3 exerted an opposite effect. In particular, co-transfection studies showed that Sp1 could significantly abolish the KLF4-induced transactivation of the CLMP gene, suggesting that KLF4 and Sp1 might compete for the same binding site on the CLMP promoter. Taken together, this differential interaction of the transcription factors, GATA-1, GATA-6, KLF4, Sp1, and Sp3, in CLMP gene expression might provide a precise machinery in regulating Sertoli cell tight junction dynamics.

Lumbar disc degeneration is linked to a carbohydrate sulfotransferase 3 variant.

Lumbar disc degeneration (LDD) is associated with both genetic and environmental factors and affects many people worldwide. A hallmark of LDD is loss of proteoglycan and water content in the nucleus pulposus of intervertebral discs. While some genetic determinants have been reported, the etiology of LDD is largely unknown. Here we report the findings from linkage and association studies on a total of 32,642 subjects consisting of 4,043 LDD cases and 28,599 control subjects. We identified carbohydrate sulfotransferase 3 (CHST3), an enzyme that catalyzes proteoglycan sulfation, as a susceptibility gene for LDD. The strongest genome-wide linkage peak encompassed CHST3 from a Southern Chinese family­based data set, while a genome-wide association was observed at rs4148941 in the gene in a meta-analysis using multiethnic population cohorts. rs4148941 lies within a potential microRNA-513a-5p (miR-513a-5p) binding site. Interaction between miR-513a-5p and mRNA transcribed from the susceptibility allele (A allele) of rs4148941 was enhanced in vitro compared with transcripts from other alleles. Additionally, expression of CHST3 mRNA was significantly reduced in the intervertebral disc cells of human subjects carrying the A allele of rs4148941. Together, our data provide new insights into the etiology of LDD, implicating an interplay between genetic risk factors and miRNA.

Structure and biology of the intervertebral disk in health and disease.

The intervertebral disks along the spine provide motion and protection against mechanical loading. The 3 structural components, nucleus pulposus, annulus fibrosus, and cartilage endplate, function as a synergistic unit, though each has its own role. The cells within each of these components have distinct origins in development and morphology, producing specific extracellular matrix proteins that are organized into unique architectures fit for intervertebral disk function. This article focuses on various aspects of intervertebral disk biology and disruptions that could lead to diseases such as intervertebral disk degeneration.

Nectin-2 expression in testicular cells is controlled via the functional cooperation between transcription factors of the Sp1, CREB, and AP-1 families.

Nectin-2, a major protein component of the adherens junctions (AJs), is found between Sertoli cells and germ cells in the seminiferous epithelium. Recent studies have shown that the expression of nectin-2 gene in testis is crucial to maintain normal spermatogenesis since male knockout mice lacking nectin-2 gene are sterile and possess morphologically abnormal spermatozoa. However, the molecular mechanisms governing its basal transcription remain poorly understood. By the use of Sertoli and germ cell-lines (TM4 and GC-2spd(ts) cells, respectively) in transient transfection studies, we showed that the minimal mouse nectin-2 promoter was located between nucleotides -316 and -211 (relative to the translation start site). Two putative Sp1 motifs and one each of the CRE, AP1, and AP2 motifs were identified within this region. Mutational studies showed that these two Sp1 motifs cooperated synergistically with the CRE motif, but not the AP1 and AP2 motifs, to regulate nectin-2 gene transcription in both TM4 and GC-2spd(ts) cells. By EMSAs, we found that an AP-1 consensus sequence was able to inhibit DNA-protein complex formation with the CRE motif, suggesting an interaction between the AP-1 transcription factor (c-Jun) and CREB within the CRE motif. Overexpressions of CREB and c-Jun, but not c-Fos, also significantly increased the promoter activity, which suggests that CREB and c-Jun are the crucial transcription factors involved in regulating nectin-2 gene transcription. Chromatin immunoprecipitation assay has shown that, in vivo, CREB, c-Jun, and Sp1 family proteins are bound to the mouse nectin-2 promoter. Analysis of the staged tubules has confirmed that the cyclic expressions of CREB and nectin-2 coincide with the event of adherens junction restructuring between Sertoli cells and germ cells. The cross-talk between CREB, c-Jun, and Sp1 family protein is believed to be a major transcription machinery to drive nectin-2 expression in Sertoli cells.