TACC3 expression is tightly regulated during early differentiation.

Transforming acidic coiled-coil (TACC) proteins are hypothesized to play a role in normal cellular growth and differentiation and to be involved in centrosomal microtubule stabilization. Our current studies aim to delineate the expression pattern of TACC3 protein during cellular differentiation and in a variety of normal human tissues. TACC3 is known to be upregulated in differentiating erythroid progenitor cells following treatment with erythropoietin and is required for replication of hematopoietic stem cells. However, we demonstrate that a dramatic upregulation of TACC3 also occurs during the early differentiation of NIH 3T3-L1 cells into adipocytes and PC12 cells into neurons, indicating that TACC3 mediates cellular differentiation in several cell types. Using real-time PCR, we quantitated the mRNA levels of TACC3 compared to TACC1 and TACC2 in various human adult tissues. We observed the highest expression of TACC3 mRNA in testis, spleen, thymus and peripheral blood leukocytes, all tissues undergoing high rates of differentiation, and a lower level of expression in ovary, prostate, pancreas, colon, small intestine, liver and kidney. In contrast, TACC1 and TACC2 mRNA levels are more widespread. By immunohistochemistry, we confirm that the TACC3 protein localizes to differentiating cell types, including spermatocytes, oocytes, epithelial cells, bone marrow cells and lymphocytes. Thus, these observations are concordant with a basic role for TACC3 during early stages of differentiation in normal tissues.

Sptrx-2, a fusion protein composed of one thioredoxin and three tandemly repeated NDP-kinase domains is expressed in human testis germ cells.

BACKGROUND: Thioredoxins (Trx) are small redox proteins that function as general protein disulphide reductases and regulate several cellular processes such as transcription factor DNA binding activity, apoptosis and DNA synthesis. In mammalian organisms, thioredoxins are generally ubiquitously expressed in all tissues, with the exception of Sptrx-1 which is specifically expressed in sperm cells. RESULTS: We report here the identification and characterization of a novel member of the thioredoxin family, the second with a tissue-specific distribution in human sperm, termed Sptrx-2. The Sptrx-2 ORF (open reading frame) encodes for a protein of 588 amino acids with two different domains: an N-terminal thioredoxin domain encompassing the first 105 residues and a C-terminal domain composed of three repeats of a NDP kinase domain. The Sptrx-2 gene spans about 51 kb organized in 17 exons and maps at locus 7p13-14. Sptrx-2 mRNA is exclusively expressed in human testis, mainly in primary spermatocytes, while Sptrx-2 protein expression is detected from the pachytene spermatocytes stage onwards, peaking at round spermatids stage. Recombinant full-length Sptrx-2 expressed in bacteria displayed neither thioredoxin nor NDP kinase enzymatic activity. CONCLUSIONS: The sperm specific expression of Sptrx-2, together with its chromosomal assignment to a position reported as a potential locus for flagellar anomalies and male infertility phenotypes such as primary ciliary dyskinesia, suggests that it might be a novel component of the human sperm axonemal organization.

Isolation and characterization of AINT: a novel ARNT interacting protein expressed during murine embryonic development.

Basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) proteins form dimeric transcription factors to mediate diverse biological functions including xenobiotic metabolism, hypoxic response, circadian rhythm and central nervous system midline development. The Ah receptor nuclear translocator protein (ARNT) plays a central role as a common heterodimerization partner. Herein, we describe a novel, embryonically expressed, ARNT interacting protein (AINT) that may be a member of a larger coiled-coil PAS interacting protein family. The AINT C-terminus mediates interaction with the PAS domain of ARNT in yeast and interacts in vitro with ARNT and ARNT2 specifically. AINT localizes to the cytoplasm and overexpression leads to non-nuclear localization of ARNT. A dynamic pattern of AINT mRNA expression during embryogenesis and cerebellum ontogeny supports a role for AINT in development.

Suspension-mediated induction of Hepa 1c1c7 Cyp1a-1 expression is dependent on the Ah receptor signal transduction pathway.

We have recently demonstrated that release of normal human epithelial cells from cell-substratum and/or cell-cell adhesion generates cellular signals that induce the expression of CYP1A1 in the absence of xenobiotic polycyclic aromatic hydrocarbons (Sadek, C. M., and Allen-Hoffmann, B. L. (1994) J. Biol. Chem. 169, 16067-16074). To directly test the involvement of the Ah receptor signal transduction pathway in CYP1A1 induction following suspension of epithelial cells, we analyzed wild-type Hepa 1c1c7 cells, a subclone of the Hepa-1c1 mouse hepatoma line, and two mutant Hepa 1c1c7 lines, Class I and Class II. Suspension of wild-type Hepa 1c1c7 cells for 4 h led to an induction of steady state levels of CYP1A1 mRNA, similar to that obtained following treatment of adherent cells with 10(-9) M 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mutants of the Hepa 1c1c7 cells defective in different aspects of the Ah receptor signal transduction pathway exhibited negligible (Class I) or no (Class II) suspension-mediated induction of CYP1A1 mRNA. Gel mobility shift analysis of nuclear extracts from suspended or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated wild-type cells showed that both treatments produced identical shifts in the mobility of an XRE-containing probe. Antibody supershift experiments confirmed that the Ah receptor was a component of the DNA-protein complex from suspended wild-type Hepa 1c1c7 cells. These data directly demonstrate that suspension of wild-type Hepa 1c1c7 cells leads to nuclear localization and activation of the Ah receptor to a DNA-binding form.

Cytochrome P450IA1 is rapidly induced in normal human keratinocytes in the absence of xenobiotics.

Cytochrome P450IA1 is a polycyclic aromatic hydrocarbon (PAH)-responsive monooxygenase enzyme with no known endogenous inducer or substrate. We investigated the effect of suspension on P450IA1 gene (CYP1A1) expression in cultured human keratinocytes without the addition of xenobiotics. To prohibit adhesion and trigger differentiation, human keratinocytes or dermal fibroblasts were suspended in medium made semisolid with methylcellulose. Following suspension, we observed dramatic increases (> 100-fold) in steady state P450IA1 mRNA in keratinocytes within 1 h; however, dermal fibroblasts were nonresponsive. This effect was not dependent on methylcellulose itself and could be achieved by suspension in medium alone or in Percoll solution. The induction of P450IA1 mRNA was independent of exogenous calcium or serum concentrations, agents commonly used to signal differentiation. Adherent keratinocytes overlaid with methylcellulose exhibited slight morphological changes accompanied by increased P450IA1 mRNA. The activity of the P450IA1 enzyme was found to parallel Northern analysis data. Changes in keratinocyte adhesion or shape also affect other PAH-responsive genes suggesting Ah receptor involvement. We report a novel mechanism for cell type-specific induction of CYP1A1 expression without the addition of xenobiotic inducers. These findings suggest a possible endogenous role for P450IA1 in stratified squamous epithelia.