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Tissue transglutaminase (transglutaminase type II) is an intracellular protein cross-linking enzyme that accumulates in connective tissue and in cells undergoing apoptosis. Retinoids regulate the transcription of the mouse tissue transglutaminase gene via activation of regulatory elements contained within 4 kilobases of the 5'-end of the gene. Co-transfection studies with retinoid receptor expression vectors in CV-1 cells demonstrated that the mouse tissue transglutaminase promoter is activated by ligand activation of either retinoic acid receptor-retinoid X receptor (RAR.RXR) heterodimers or RXR homodimers. Optimal induction is achieved with retinoid receptor panagonists; partial activation can also be achieved with either RAR-specific or RXR-specific retinoids. Retinoid-dependent activation of the tissue transglutaminase promoter depends on both a proximal regulatory region containing sequences highly conserved between the human and the mouse tissue transglutaminase promoters and a distal region that includes a 30-base pair retinoid response element (mTGRRE1). mTGRRE1 contains three hexanucleotide half-sites (two canonical and one non-canonical) in a DR7/DR5 motif that bind both RAR*RXR heterodimers and RXR homodimers. These studies suggest that retinoid-dependent expression of the mouse tissue transglutaminase gene is mediated by a versatile tripartite retinoid response element located 1.7 kilobases upstream of the transcription start site.

Tissue transglutaminase belongs to a family of calcium-dependent enzymes, the transglutaminases that catalyze the covalent cross-linking of specific proteins by the formation of epsilon (gamma-glutamyl)lysine isopeptide bonds. The goal of this study has been the isolation and characterization of the human tissue transglutaminase gene promoter. Genomic DNA clones, spanning the 5' region of the gene, were isolated and the structure of the 5'-end of the human tissue transglutaminase gene was determined. 1.74 kilobases of flanking DNA were sequenced and were found to contain a TATA box element (TATAA), a CAAT box element (GGACAAT), a series of potential transcription factor-binding sites (AP1, SP1, interleukin-6 response element), and a glucocorticoid response elements. Transient transfection experiments showed that this DNA fragment included a functional promoter, which is constitutively active in multiple cell types.

Fresh serum and retinoids induce the expression of tissue transglutaminase in cultured mouse resident peritoneal macrophages. Analogues of cyclic AMP, such as dibutyryl cyclic AMP, and agents that increase intracellular cyclic AMP levels enhance the induction. Dibutyryl cyclic AMP alone has little effect on transglutaminase expression, but it increases the sensitivity of macrophages to low concentrations of either serum or retinoic acid. Dibutyryl cyclic AMP potentiates the transglutaminase-inducing activity of both free retinoic acid and retinoic acid bound to the serum retinol-binding protein. Pretreating macrophages with dibutyryl cyclic AMP or retinoic acid does not prime the cells to respond to the other agent; instead, both agents must be present simultaneously to obtain the synergistic induction of transglutaminase. Our studies suggest that the modulation of intracellular cyclic AMP levels may have pronounced effects on retinoic acid-induced gene expression in myeloid cells.

Retinoic acid rapidly induces the accumulation of a specific enzyme, tissue transglutaminase (EC 2.3.2.13), in mouse macrophages. We have used the induction of tissue transglutaminase to study the regulation of gene expression by retinoic acid. In this study we report that pertussis toxin can inhibit retinoic acid-induced expression of tissue transglutaminase in mouse resident peritoneal macrophages. This inhibition is paralleled by the ADP-ribosylation of 41,000-dalton macrophage membrane protein.

We have purified the high molecular weight actin-binding protein, filamin from guinea pig vas deferens. We find this mammalian filamin is very similar to chicken gizzard filamin in subunit molecular weight, amnio acid composition, actin-binding properties, immunological cross-reactivity, and the ability to be phosphorylated by cyclic AMP-dependent protein kinase. Anti-filamin antibodies cross-react with a high molecular weight macrophage actin-binding protein, and with a high molecular weight protein in platelets and fibroblasts. Furthermore like filamin, these proteins are also phosphorylated and cyclic AMP stimulates their phosphorylation. Anti-filamin antibodies do not cross-react with the erythrocyte membrane protein spectrin or with high molecular weight proteins in brain extracts. We conclude that filamin from avian and mammalian smooth muscle are very similar proteins and furthermore that many, but not all, non-muscle cells contain a protein closely related to filamin.

Tissue transglutaminase accumulates rapidly and to very high levels (1-2% of cellular protein) in mouse peritoneal macrophages cultured in mouse serum. The induction is due to accelerated synthesis of the enzyme (150-fold increase) that occurs within 90 min of exposure of the cells to a heat-labile constituent of serum or plasma. The induction is reversible and is not reproduced by known activators of macrophage function such as lipopolysaccharide, muramyl dipeptide, and tuftsin. In animals, elevated levels of tissue transglutaminase are also found in inflammatory macrophages elicited by thioglycolate broth.

Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells.

The culture of peritoneal macrophages in serum-containing media induces a dramatic increase in the expression of the enzyme tissue transglutaminase. The transglutaminase-inducing activity of serum is abolished by extraction of lipids and fully restored by re-addition of physiological concentrations (1-100 nM) of trans-retinoic acid. Induction of the enzyme is detectable within a 90-min exposure of macrophages to retinoic acid and is completely blocked by actinomycin D, suggesting that the retinoid rapidly increases the rate of transglutaminase gene expression. Delipidized serum is required to elicit the transglutaminase-inducing activity of retinoic acid and this effect is decreased if the serum is depleted of the serum retinol-binding protein. Our studies suggest that retinoic acid and serum retinol-binding protein can directly regulate macrophage gene expression and specifically induce the synthesis of tissue transglutaminase.

Chicken gizzard filamin has been digested with purified Ca2+-activated protease. The subunits of (Mr = 250,000) of the protein are cleaved asymmetrically into two fragments, heavy merofilamin, Mr = 240,000, and light merofilamin, Mr = 9,500. Digestion is complete at substrate to enzyme ratios of 100:1 and requires Ca2+ concentrations in excess of 0.3 mM. Heavy merofilamin binds to F-actin as evidenced by cosedimentation with F-actin, by direct observation under the electron microscope, and by its ability to inhibit actin activation of heavy meromyosin ATPase. Heavy merofilamin does not form a gel when mixed with actin, except at very low concentrations of KCl. Thus, actin binding and gelation are separable activities of filamin. We speculate that Ca2+-stimulated proteolysis may play a role in the regulation of actin-filamin interactions.

Filamin, a protein recently identified in chicken gizzard (Wang, K., Ash, F., and Singer, S. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4483-4486), has been purified free of other components and its molecular properties have been examined. Filamin has a sedimentation constant (S020,w) of 8.86 S and a partial specific volume of 0.734 ml/g. Sedimentation equilibrium experiments give a value of 498,000 for the molecular weight of native filamin. From these data a frictional ratio of 2.32 has been calculated. On sodium dodecyl sulfate gel electrophoresis, filamin migrates as a single protein band with an estimated molecular weight of 240,000. Filamin is a soluble protein and under a variety of conditions tested does not by itself form filaments or precipitate from solution. However, filamin binds to rabbit skeletal muscle F-actin, and the complex is readily sedimented by centrifugation to yield a gelatinous pellet containing actin and filamin. These results indicate that filamin is a dimeric protein with a moderate degree of asymmetry that binds to actin. The results also suggest that the distribution of filamin in cells is derived from its interaction with polymerized actin.