Thrombospondin 1 and thrombospondin 2 are expressed as both homo- and heterotrimers.

There exist two distinct thrombospondin molecules (designated TSP1 and TSP2) which are encoded by separate genes. TSP1 is a trimeric cell surface and extracellular matrix molecule. Sequence comparison reveals that the 2 cysteines involved in interchain disulfide linkage and trimer assembly in TSP1 are conserved in TSP2 (Laherty, C. D., O'Rourke, K., Wolf, F. W., Katz, R., Seldin, M. F., and Dixit, V. M. (1992) J. Biol. Chem. 267, 3274-3281). Swiss 3T3 fibroblasts express both TSP1 and TSP2, and, therefore, an important question is whether TSP in such cells is expressed as homotrimers or as heterotrimers. We find that Swiss 3T3 cells and epithelial cells transfected with TSP expression vectors express both homo- and heterotrimeric forms of TSP. In addition, homotrimeric TSP2 has a lower affinity for heparin than homotrimeric TSP1. Thus, the heparin affinity of TSP can be modulated by the expression of TSP as homo- or heterotrimers.     

Glucocorticoid effects on newly synthesized proteins in muscle and non-muscle cells cultured from neonatal rat hearts

To analyze direct effects of steroids on the rates of synthesis (and/or degradation) of newly synthesized proteins of the rat heart, we have used high resolution two-dimensional gel electrophoresis and autoradiography. A collective steroid domain of nineteen proteins, comprising fifteen with an increased rate of synthesis and four with a decreased rate of synthesis, was consistently seen in cultures of cardiac muscle and non-muscle cells from neonatal rats following 24 h incubation with 10(-7) dexamethasone. Similarly, incubation with 10(-7) M sex steroids, mineralocorticoids, and other glucocorticoids including the highly selective compound RU26988, established the glucocorticoid-specificity of the response. Different subsets of this glucocorticoid domain were seen for collagenase- or trypsin-dispersed primary cultures of cardiac muscle and non-muscle cells or for passaged cultures of cardiac non-muscle cells. Six polypeptides were consistently induced in all cardiac cultures, regardless of cell morphology. Two polypeptides were consistently induced only in those cultures containing cardiac non-muscle cells, whereas protein l, of identical Mr(approximately 52K) and pI (approximately 5.3) to desmin, was induced only in cultures of spontaneously contractile cardiac muscle cells. The glucocorticoid domain proteins described herein represent direct steroid effects on cardiac cells and are therefore candidate mediators of physiological glucocorticoid effects on, for example, differentiation and contractility.     

Multiple growth hormone-binding proteins are expressed on insulin-producing cells

The insulin-producing rat islet tumor cell line, RIN-5AH, expresses somatogen binding sites and responds to GH by increased proliferation and insulin production. Affinity cross-linking shows that RIN-5AH cells contain two major GH-binding subunits of Mr 100-130K (110K), which appear to exist as disulfide-linked multimers of Mr 270-350K (300K). In addition, a minor Mr 180K GH-binding protein is identified which does not appear to be associated with other proteins by disulfide bridges. A plasma membrane-enriched fraction accounts for 86% of the RIN-cell GH-binding activity while cytosol and intracellular organelles are low in GH-binding activity. The plasma membrane-bound activity is soluble in Triton X-100 with intact hormone binding characteristics. The apparent KD in detergent solution is estimated to 18 ng/ml (8 x 10(-10) M). 125I-hGH-affinity cross-linking to intact and detergent-solubilized membranes as well as hGH-affinity purified protein reveals labeled proteins of Mr 180K and Mr 285-350K. In contrast to the cross-linked Mr 300K complexes of intact cells those of disintegrated cellular material are resistant to reduction with dithiothreitol, and it is speculated that this is due to intersubunit cross-linking of the disulfide-linked Mr 110K GH-binding subunits. The GH-binding proteins are purified approximately 100-fold by one cycle of hGH-affinity chromatography and five major proteins of Mr 180K, 94K, 86K, 64K, and 54K are identified by silver staining in the purified fraction. It is concluded that the RIN-5AH cells have multiple GH-binding proteins which may mediate signals for either proliferation and/or insulin production.     

Distribution of actin and myosin in muscle and non-muscle cells

Summary Specific anti-actin and anti-myosin antibodies were shown to react in single and double immunofluorescence sandwich tests with identical sites in non-muscle cells in frozen sections of tissues and in cultured cells. In tissues, both antibodies reacted with liver cell membranes, parts of renal glomeruli, brush borders and peritubular fibrils of renal tubules, brain synaptic junctions, and membranes of lymphoid cells in thymic medulla, lymph nodes and spleen. Both antibodies reacted strongly with long parallel cytoplasmic fibrils in cultured fibroblasts, and with disrupted fibrils in cytochalasin-B treated cells. In neuroblastoma cells both antibodies gave prominent staining of growth cones and microspikes. The observation that the distribution of myosin parallels that of actin in non-muscle cells argues strongly in favour of a functional interaction between the two molecules in the generation of contractile activity in nonmuscle cells.The authors thank Dr. M. Owen, National Institute of Medical Research, Mill Hill, for the gift of rabbit anti-actin antibodyOn sabbatical leave from Monash University, and supported by a Commonwealth Medical FellowshipThe Brompton Hospital, London     

The LIM proteins FHL1 and FHL3 are expressed differently in skeletal muscle

We have determined the complete mRNA sequence of FHL3 (formerly SLIM2). We have confirmed that it is a member of the family of LIM proteins that share a similar secondary protein structure, renamed as Four-and-a-Half-LIM domain (or FHL) proteins in accordance with this structure. The "half-LIM" domain is a single zinc finger domain that may represent a subfamily of LIM domains and defines this particular family of LIM proteins. The distribution of FHL mRNA expression within a variety of murine tissues is complex. Both FHL1 and FHL3 were expressed in a number of skeletal muscles while FHL2 was expressed at high levels in cardiac muscle. Localisation of FHL3 to human chromosome 1 placed this gene in the proximity of, but not overlapping with, alleles associated with muscle diseases. FHL1 and FHL3 mRNAs were reciprocally expressed in the murine C2C12 skeletal muscle cell line and this suggested that the pattern of expression was linked to key events in myogenesis.     

Non-identity of muscle and non-muscle actins.

Tryptic peptide maps of actin prepared from chicken muscle and chick brain appear to be very similar, but not identical. Brain actin lacks at least one peptide found in muscle actin and its fingerprint contains approximately six additional peptides. Whether these differences between muscle and cytoplasmic actins are due to their synthesis from different genes or to post-translational modification is not yet known.     

Dynamics of the endoplasmic reticulum in living non-muscle and muscle cells

The dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3'-dihexyloxacarbocyanine iodide. Two types of arrays characterize the continuous ER system in the non-muscle PtK2 cell: 1) a lacy network of irregular polygons and 2) long strands of ER that are found aligned along stress fibers. In cross-striated myotubes there was a periodic localization of fluorescence over each I-band corresponding to the positions of the terminal cisternae of the sarcoplasmic reticulum (SR). In contrast to the arrangement in muscle cells, the alignment of the long strands of ER alon stress fibers showed no strict periodicity that could be correlated with the sarcomeric units of the stress fibers. The ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the endoplasmic reticulum and sarcoplasmic reticulum, respectively. Observations of vitally stained PtK2 cells at 1 to 2 minute intervals using low light level video cameras and image processing techniques enabled us to see the polygonal ER units form and undergo changes in their shapes. During cell division, the ER, rhodamine 123-stained mitochondria, and phagocytosed fluorescent beads were excluded from the mitotic spindle while soluble proteins were not. No obvious concentration or alignment of membranes could be found associated with the contractile proteins in the cleavage furrow. After completion of cell division there was a redeployment of the ER network in each daughter cell.     

The MEF2A and MEF2D function as scaffold proteins that interact with HDAC1 or p300 in SOD3 expression in THP-1 cells

Superoxide dismutase 3 (SOD3) is a SOD isozyme and plays a key role in extracellular redox homeostasis. We previously demonstrated that histone acetylation is involved in 12-O-tetra-decanoylphorbol-13-acetate (TPA)-elicited SOD3 expression in human monocytic THP-1 cells; however, the molecular mechanisms responsible for its expression have not yet been elucidated in detail. The results of the present study demonstrated that the binding of histone deacetylase 1 (HDAC1) to the SOD3 promoter region contributed to SOD3 silencing in basal THP-1 cells. On the other hand, the dissociation of HDAC1 from the SOD3 promoter region and the enrichment of p300, a histone acetyltransferase (HAT), within that region were observed in TPA-induced THP-1 cells. Myocyte enhancer factor 2 (MEF2) functions as a scaffold protein that interacts with histone deacetylases (HDAC) or HAT and regulates gene expression. The present results showed that the MEF2A and MEF2D function as mediators for TPA-elicited SOD3 expression by interacting with HDAC or p300. Additionally, the knockdown of MEF2A or MEF2D in human skin fibroblasts suppressed SOD3 expression at the mRNA and protein levels. Our results provide an insight into epigenetic regulation of redox gene expression, and may ultimately contribute to suppressing the progression of tumours and vascular diseases.     

VAMP2 is expressed in muscle satellite cells and up-regulated during muscle regeneration

Membrane trafficking is one of the most important mechanisms involved in the establishment and maintenance of the forms and functions of the cell. However, it is poorly understood in skeletal muscle cells. In this study, we have focused on vesicle-associated membrane proteins (VAMPs), which are components of the vesicle docking and fusion complex, and have performed immunostaining to investigate the expression of VAMPs in rat skeletal muscle tissue. We have found that VAMP2, but not VAMP1 or VAMP3, is expressed in satellite cells. VAMP2 is also expressed in myofibers in the soleus muscle and nerve endings. This is consistent with previous studies in which VAMP2 has been shown to regulate GLUT4 trafficking in slow-twitch myofibers in soleus muscle and neurotransmitter release in nerve endings. As satellite cells are quiescent myogenic cells, the expression of VAMP2 has further been examined in regenerating muscles after injury by the snake venom, cardiotoxin; we have observed enhanced expression of VAMP2 in immature myotubes with a peak at 3 days after injury. Our findings suggest that VAMP2 plays roles in quiescent satellite cells and is involved in muscle regeneration. The nature of the material transported in the VAMP2-bearing vesicles in satellite cells and myotubes is still under investigation. This work was supported by a research grant (17A-10) for nervous and mental disorders from the Ministry of Health, Labor, and Welfare of Japan, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

Phosphorylation of myosin in non-muscle and smooth muscle cells. Possible rules and evolutionary trends

Reversible phosphorylation of myosin subunits is observed in almost all eukaryotic cells. The data concerning sites and effects of phosphorylation on actin-activated ATPase activity of myosin and on its filament formation are described. These observations are discussed in terms of possible evolutionary trends and rules which may govern the process of myosin phosphorylation.     

Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle

Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca2+ entry triggered by depletion of Ca2+ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca2+ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca2+ signaling in distal PASMCs, we measured [Ca2+]i by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca2+-free media containing cyclopiazonic acid (10 microM) and nifedipine (5 microM) to deplete ER Ca2+ stores and block voltage-dependent Ca2+ channels, restoration of extracellular Ca2+ (2.5 mM) caused marked increases in [Ca2+]i whereas MnCl2 (200 microM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl3, and NiCl2, blocked CCE at concentrations that did not alter Ca2+ responses to 60 mM KCl (IC50 6.3, 40.4, and 191 microM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, -4, and -6, but not TRPC2, -3, -5, or -7. Our results suggest that CCE through TRPC-encoded Ca2+ channels could contribute to Ca2+ signaling in myocytes from distal intrapulmonary arteries.     

Zinedin, SG2NA, and striatin are calmodulin-binding, WD repeat proteins principally expressed in the brain

Striatin is an intracellular protein characterized by four protein-protein interaction domains, a caveolin-binding motif, a coiled-coil structure, a calmodulin-binding domain, and a WD repeat domain, suggesting that it is a signaling or a scaffold protein. Down-regulation of striatin, which is expressed in a few subsets of neurons, impairs the growth of dendrites as well as rat locomotor activity (Bartoli, M., Ternaux, J. P., Forni, C., Portalier, P., Salin, P., Amalric, M., and Monneron, A. (1999) J. Neurobiol. 40, 234-243). Zinedin, a "novel" protein described here, and SG2NA share with striatin identical protein-protein interaction domains and the same overall domain structure. A phylogenetic analysis supports the hypothesis that they constitute a multigenic family deriving from an ancestral gene. DNA probes and antibodies raised against specific domains of each protein showed that zinedin is mainly expressed in the central nervous system, whereas SG2NA, of more widespread occurrence, is mainly expressed in the brain and muscle. All three proteins are both cytosolic and membrane-bound. All three bind calmodulin in the presence of Ca(2+). In rat brain, SG2NA and striatin are generally not found in the same neurons. Both localize to the soma and dendrites, suggesting that they share a similar type of addressing and closely related functions.