Previous studies on the promoter function of the human C-type natriuretic peptide (CNP) gene have revealed the existence of two GC-rich cis elements essential for gene transcription in rat pituitary-derived GH3 cells. To isolate transcription factors that bind to those GC-rich elements, a lambda ZAP cDNA library derived from GH3 cells was screened. Several positive clones with specific binding abilities were obtained; one is identical as TSC-22, a speculated transcriptional modulator stimulated by transforming growth factor beta (TGF-beta) of unknown function. TSC-22 significantly enhances CNP promoter activity in GH3 cells. In adults, human TSC-22 mRNA is highly expressed in brain, lung and heart. TSC-22 gene expression in GH3 and human aortic endothelial cells is stimulated by cytokines, including TGF-beta, in concert with the CNP mRNA increase. These results suggest that TSC-22 is a transcriptional regulator of the CNP gene and transmits signals from cytokines, such as TGF-beta, to CNP gene expression (Ohta, 1996).

The molecular mechanisms underlying the pleiotropic effects of FSH were investigated by screening a plasmid cDNA library for clones hybridizing to FSH-regulated RNAs from FSH-treated Sertoli cells. One clone encodes the rat homolog of transforming growth factor-beta 1-stimulated clone 22 (TSC-22), which contains a putative leucine zipper region. Regulation of rat TSC-22 mRNA was analyzed in primary Sertoli cell cultures. TSC-22 mRNA transiently increases 4-fold in the presence of FSH, reached maximal levels at 3 h, and returns to prestimulation levels by 12 h. The FSH-stimulated increase is independent of protein synthesis because it occurs in the presence of cycloheximide and FSH. TSC-22 mRNA is detected in all tissues examined in male and female rats; the highest levels in the 16-day animal were observed in the testis, ovary, uterus, and lung. Testicular 1.8-kilobase (kb) TSC-22 mRNA decreases by 50% from 14 to 60 days of age. A 5-kb transcript becomes detectable by 30 days and decreases after 50 days of age. Ovarian 1.8-kb TSC-22 transcript levels increased about 2-fold during the same maturation period (Hamil, 1997).

A gene sequence (TSC-22) that is induced by transforming growth factor (TGF) beta 1 was isolated by differential screening of a cDNA library constructed from poly(A)+ RNA of mouse osteoblastic cells treated with TGF-beta. TSC-22 gene expression is transcriptionally activated by TGF-beta 1. It is also induced by phorbol 12-myristate 13-acetate, serum, cholera toxin, or dexamethasone, but not appreciably by epidermal growth factor. Its induction is rapid and transient, reaching a peak 2 h after TGF-beta 1 treatment, and is resistant to cycloheximide like that of c-jun. The nucleotide sequences of TSC-22 cDNA show no homology with any known gene sequence. The open reading frame and in vitro translation product indicate that the gene encodes a polypeptide of 143 amino acids with a molecular mass of 18 kDa that contains a putative leucine zipper structure. Polyclonal antibody was raised against TSC-22 protein expressed in Escherichia coli cells; the antibody detects a 18-kDa protein in both the cytoplasmic and nuclear fractions of cells. These results indicate that the TSC-22 gene is a new member of the family of early response genes, and encodes a small polypeptide that is a putative transcriptional regulator (Shibanuma, 1992).

Human gastric carcinoma cell line HSC-39 has been shown to undergo apoptotic cell death in response to treatment with transforming growth factor beta1 (TGF-beta1). To understand better the cell death mechanism in this TGF-beta1-mediated apoptosis, the effect of the expression of TGF-beta-stimulated clone 22 (TSC-22) on cell death was investigated. TGF-beta1 induces TSC-22 gene expression in HSC-39 cells only when the cells have previously been adapted to the serum-free culture conditions required to undergo TGF-beta1-mediated apoptosis. HSC-39 cells transfected with a TSC-22 expression vector show a significant decrease in cell viability compared with those transfected with a control vector. The cellular events characteristic of apoptosis, chromatin condensation and DNA fragmentation, are observed only in cells transfected with a TSC-22 expression vector. On immunostaining of the transfected cells, almost every cell that expresses TSC-22 tagged with influenza virus hemagglutinin exhibits the morphology of an apoptotic cell. Partial protection from the cell death effect of TGF-beta1 on HSC-39 cells is observed when cells are treated with acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspart-1-al (Ac-DEVD-CHO, an inhibitor specific for CPP32-type protease). Protection against cell death by the transfection of a TSC-22 expression vector is also offered by Ac-DEVD-CHO addition. These results suggest that TSC-22 elicits the apoptotic cell death of human gastric carcinoma cells through the activation of CPP32-like protease and mediates the TGF-beta1 signaling pathway to apoptosis (Ohta, 1997).

TGF-beta-stimulated clone-22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved during evolution. Two homologs are known that share a similar leucine zipper domain and another conserved domain (designated the TSC box). Only limited data are available on the function of TSC-22 and its homologs. TSC-22 is transcriptionally up-regulated by many different stimuli, including anti-cancer drugs and growth inhibitors; recent data suggest that TSC-22 may play a suppressive role in tumorigenesis. TSC-22 forms homodimers via its conserved leucine zipper domain. Using a yeast two-hybrid screen, a TSC-22 homolog (THG-1) is found to be a heterodimeric partner. The presence of two more mammalian family members with highly conserved leucine zippers and TSC boxes is reported. Interestingly, both TSC-22 and THG-1 have transcriptional repressor activity when fused to a heterologous DNA-binding domain. The repressor activity of TSC-22 appears sensitive for promoter architecture, but not for the histone deacetylase inhibitor trichostatin A. Mutational analysis shows that this repressor activity resides in the non-conserved regions of the protein and is enhanced by the conserved dimerization domain. These results suggest that TSC-22 belongs to a family of leucine zipper-containing transcription factors that can homodimerize and heterodimerize with other family members and that at least two TSC-22 family members may be repressors of transcription (Kester, 1999).

The 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9-46), were synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicate that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9-46), it has been concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein reveals that the left-handed super helical structure of the leucine zipper type sequence is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids, the structure of pDIP is stabilized by the formation of interhelical salt bridges. This result has been confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed (Seidel, 1997)

The leucine zipper transcription factor TSC-22 (TGF-beta1 Stimulated Clone-22) was first isolated from a mouse osteoblast cell line as an immediate-early target gene of TGF-beta1. However, work with other cell lines, as well as with a Drosophila homolog, bunched, suggests that it is an effector gene of various growth factors and potentially involved in the integration of multiple extracellular signals. Throughout mouse embryogenesis TSC-22 is expressed in a dynamic pattern. Although early TSC-22 expression is ubiquitous in 6.5 day embryos, as development proceeds TSC-22 expression is upregulated at sites of epithelial-mesenchymal interactions such as the limb bud, tooth primordiun, hair follicle, kidney, lung, and pancreas. TSC-22 is also expressed in many neural crest-derived tissues including the mesenchyme of the branchial arches, the cranial, dorsal root, and sympathetic ganglia, as well as the facial cartilage and bone. Other areas of expression are the otic and optic vesicles, the heart, and cartilage and bone forming regions throughout the embryo (Dohrmann, 1999).

This study was undertaken to clarify the molecular mechanism of the effect of a new anti-cancer drug, vesnarinone, on a human salivary gland cancer cell line, TYS. TSC-22cDNA was isolated as a vesnarinone-inducible gene from a cDNA library constructed from vesnarinone-treated TYS cells. TSC-22 was originally reported as a transforming growth factor (TGF)-beta-inducible gene. The expression of TSC-22 is up-regulated within a few hours after treatment with vesnarinone and continues for 3 days. The level of TSC-22 mRNA in TYS cells continuously increases until the cells reach confluence. Furthermore, the induction of TSC-22 by vesnarinone is inhibited by treatment with cycloheximide. When cells are treated with an antisense oligonucleotide against TSC-22 mRNA under quiescent conditions, the antisense oligonucleotide stimulates the growth of TYS cells; however, under growing conditions the antisense oligonucleotide does not affect cell growth. Furthermore, the antisense oligonucleotide suppresses the antiproliferative effect of vesnarinone. These results suggest that TSC-22 may be a negative growth regulator and may play an important role in the antiproliferative effect of vesnarinone (Kawamata, 1998).

Mesenchymal stem cells have the potential to differentiate into various cell lineages, including adipocytes and osteoblasts. The induction of adipocyte differentiation by glucocorticoids (GCs) not only causes the accumulation of fat cells in bone marrow, but also depletes the supply of osteoblasts for new bone formation, thus leading to osteoporosis. A GC-induced leucine-zipper protein (GILZ) antagonizes adipocyte differentiation. GILZ binds to a tandem repeat of CCAAT/enhancer-binding protein (C/EBP) binding sites in the promoter of the gene encoding peroxisome-proliferator-activated receptor-gamma2 (PPAR-gamma2), and inhibits its transcription as a sequence-specific transcriptional repressor. Ectopic expression of GILZ blocks GC-induced adipocyte differentiation. Furthermore, adipogenic marker genes (for example, those encoding PPAR-gamma2, C/EBP-alpha, lipoprotein lipase and adipsin) are also inhibited by GILZ. These results reveal a novel GC antagonistic mechanism that has potential therapeutic applications for the inhibition of GC-induced adipocyte differentiation (Shi, 2003).

Peroxisome proliferator-activated receptor gamma (PPARgamma) and transforming growth factor-beta (TGF-beta) are key regulators of epithelial cell biology. However, the molecular mechanisms by which either pathway induces growth inhibition and differentiation are incompletely understood. Transforming growth factor-simulated clone-22 (TSC-22) has been identified as a target gene of both pathways in intestinal epithelial cells. TSC-22 is member of a family of leucine zipper containing transcription factors with repressor activity. Although little is known regarding its function in mammals, the Drosophila homolog of TSC-22, bunched, plays an essential role in fly development. The ability of PPARgamma to induce TSC-22 was not dependent on an intact TGF-beta1 signaling pathway and is specific for the gamma isoform. Localization studies revealed that TSC-22 mRNA is enriched in the postmitotic epithelial compartment of the normal human colon. Cells transfected with wild-type TSC-22 exhibit reduced growth rates and increased levels of p21 compared with vector-transfected cells. Furthermore, transfection with a dominant negative TSC-22 in which both repressor domains were deleted is able to reverse the p21 induction and growth inhibition caused by activation of either the PPARgamma or TGF-beta pathways. These results place TSC-22 as an important downstream component of PPARgamma and TGF-beta signaling during intestinal epithelial cell differentiation (Gupta, 2003).

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages, including osteoblasts and adipocytes. Glucocorticoid-induced leucine zipper (GILZ) inhibits peroxisome proliferator-activated receptor gamma-2 (Ppargamma2) expression and blocks adipocyte differentiation. This study shows that overexpression of GILZ in mouse MSCs, but not MC3T3-E1 osteoblasts, increases alkaline phosphatase activity and enhances mineralized bone nodule formation, whereas knockdown of Gilz reduces MSC osteogenic differentiation capacity. Consistent with these observations, real-time reverse transcription-PCR analysis showed that both basal and differentiation-induced transcripts of the lineage commitment gene Runx2/Cbfa1, as well as osteoblast differentiation marker genes including alkaline phosphatase, type I collagen, and osteocalcin, are all increased in GILZ-expressing cells. In contrast, the mRNA levels of adipogenic Ppargamma2 and C/ebpalpha are significantly reduced in GILZ-expressing cells under both osteogenic and adipogenic conditions. Together, these results demonstrate that GILZ functions as a modulator of MSCs and that overexpression of GILZ shifts the balance between osteogenic and adipogenic differentiation of MSCs toward the osteogenic pathway. These data suggest that GILZ may have therapeutic value for stem cell-based therapies of metabolic bone diseases, such as fracture repair (Zhang, 2008).

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