Plasminogen activator activity in differentiating rat myoblasts

Summary Primary cultures of skeletal muscle cells secrete plasminogen activator (PA) activity to the conditioning medium and display membrane-bound PA. Growth of these cells in culture in presence of 10^-7 M dexamethasone resulted in a marked reduction of the membranal and secreted PA activity. The hormone also reduced cytosolic creative phosphokinase (CPK) activity and cytosolic protein content. However, cell viability and their ability to undergo fusion were uneffected. The extent of hormone-induced reduction in PA activity depended on the time and extend of exposure. Maximal suppression was obtained by exposing the cells to dexamethasone during the first 4 days of culture. The medium conditioned with dexamethasone-treated cells did not inhibit plasmin, endogenous PA or exogenous PA. Exposure of the conditioned medium from hormone-treated cells to sodium dodecyl sulphate (SDS) or trypsin restored the activity to values observed in media from cells not exposed to the hormone. Acidification of the medium failed to reactivate the enzyme. The myogenic cell line L-8 also displayed membrane-bound PA activity, which was of a comparable magnitude in both fusing and non-fusing L-8 cells. However, in contrast to the primary cultures, exposure of L-8 cells to dexamethasone had no effect on their PA activity whether studied under conditions which allowed or prohibited fusion. The present findings imply that PA has no conducive role in the process of fusion associated with maturation of skeletal muscle cells.Abbreviations CPK Creative phosphokinase - PA Plasminogen activator - PBS Phosphate buffered saline - SDS Sodium dodecyl sulphate - TCA Trichloroacetic acid     

Regulation of creatine kinase induction in differentiating mouse myoblasts.

The regulation of creatine kinase (CK) induction during muscle differentiation was analyzed with MM14 mouse myoblasts. These cells withdraw from the cell cycle and commit to terminal differentiation when fed with mitogen-depleted medium. Myoblasts contained trace amounts of an isozyme of brain CK (designated BB-CK), but differentiation was accompanied by the induction of two other isozymes of muscle and brain CKs (designated MM-CK and MB-CK). Increased CK activity was detectable within 6 h of mitogen removal, 3 h after the first cells committed to differentiation and 6 h before fusion began. By 48 h, MM-CK activity increased more than 400-fold, MB-CK activity increased more than 150-fold, and BB-CK activity increased more than 10-fold. Antibodies prepared against purified mouse MM-CK cross-reacted with muscle and brain CKs (designated M-CK and B-CK, respectively) from a variety of species and were used to demonstrate that the increase in enzymatic activity was paralleled by an increase in the protein itself. CK antibodies were also used to aid in identifying cDNA clones to M-CK. cDNA sequences which corresponded to protein-coding regions cross-hybridized with B-CK mRNA; however, a subclone containing the 3'-nontranslated region was unique and was used to quantitate M-CK mRNA levels during myoblast differentiation. M-CK mRNA was not detectable in myoblasts, but within 5 to 6 h of mitogen withdrawal (6 to 7 h before fusion begins) it accumulated to about 30 molecules per cell. By 24 h, myotubes contained approximately 1,100 molecules per nucleus of M-CK mRNA.     

Pax3/7BP is a Pax7- and Pax3-binding protein that regulates the proliferation of muscle precursor cells by an epigenetic mechanism

In mouse skeletal muscles, Pax7 uniquely marks muscle satellite cells and plays some important yet unknown functions at the perinatal stage. To elucidate its in vivo functions, we initiated a yeast two-hybrid screening to look for Pax7-interacting proteins and identified a previously uncharacterized Pax7- and Pax3-binding protein (Pax3/7BP). Pax3/7BP is a ubiquitously expressed nuclear protein, enriched in Pax7+ muscle precursor cells (MPCs), and serves as an indispensable adaptor for Pax7 to recruit the histone 3 lysine 4 (H3K4) methyltransferase (HMT) complex by bridging Pax7 and Wdr5. Knockdown of Pax3/7BP abolished the Pax3/7-associated H3K4 HMT activity and inhibited the proliferation of Pax7+ MPCs from young mice both in culture and in vivo. Id3 and Cdc20 were direct target genes of Pax7 and Pax3/7BP involved in the proliferation of Pax7+ MPCs. Collectively, our work establishes Pax3/7BP as an essential adaptor linking Pax3/7 with the H3K4 HMT to regulate the proliferation of MPCs.     

Regulation of Pax3 by proteasomal degradation of monoubiquitinated protein in skeletal muscle progenitors

Pax3 and Pax7 play distinct but overlapping roles in developmental and postnatal myogenesis. The mechanisms involved in the differential regulation of these highly homologous proteins are unknown. We present evidence that Pax3, but not Pax7, is regulated by ubiquitination and proteasomal degradation during adult muscle stem cell activation. Intriguingly, only monoubiquitinated forms of Pax3 could be detected. Mutation of two specific lysine residues in the C-terminal region of Pax3 reduced the extent of its monoubiquitination and susceptibility to proteasomal degradation, whereas introduction of a key lysine into the C-terminal region of Pax7 rendered that protein susceptible to monoubiquitination and proteasomal degradation. Monoubiquitinated Pax3 was shuttled to the intrinsic proteasomal protein S5a by interacting specifically with the ubiquitin-binding protein Rad23B. Functionally, sustained expression of Pax3 proteins inhibited myogenic differentiation, demonstrating that Pax3 degradation is an essential step for the progression of the myogenic program. These results reveal an important mechanism of Pax3 regulation in muscle progenitors and an unrecognized role of protein monoubiquitination in mediating proteasomal degradation.     

Protein kinase and cyclic AMP levels in differentiating myoblasts are altered by extracellular calcium

Protein kinase specific activities and cyclic AMP levels show a similar pattern of response, when the Ca²⁺ concentration is altered in the culture medium of differentiating chick skeletal muscle cells; an increase at intermediate Ca²⁺ concentrations (0.05–0.2mM), followed by a decrease at higher concentrations (2mM). Effects of Ca²⁺ on protein kinase appear to be on cyclic AMP-independent enzymes in both nucleus and cytoplasm, and are quite the reverse of Ca²⁺ effects on the muscle-specific enzyme, creatine kinase.     

A study of protein profile changes in differentiating human myoblasts

The changes in the protein profile in cultured human myoblasts after induction of differentiation were studied by proteomic techniques (a combination of O’Farrell two-dimensional electrophoresis and subsequent protein identification by MALDI-TOF MS and MS/MS analyses). Forty-one proteins have been identified, 25 of which were present in both proliferating and differentiating myoblasts, which allows them to be considered as myoblast housekeeping proteins. The changes in the distribution of some isoforms of tropomyosins, S100 proteins, cofilin, etc. have been revealed. The possible role of these changes in the cell protein profile in the realization of the program of skeletal muscle cell differentiation is discussed.     

The proteasome is responsible for caspase-3-like activity during xylem development

Xylem development is a process of xylem cell terminal differentiation that includes initial cell division, cell expansion, secondary cell wall formation and programmed cell death (PCD). PCD in plants and apoptosis in animals share many common characteristics. Caspase-3, which displays Asp-Glu-Val-Asp (DEVD) specificity, is a crucial executioner during animal cells apoptosis. Although a gene orthologous to caspase-3 is absent in plants, caspase-3-like activity is involved in many cases of PCD and developmental processes. However, there is no direct evidence that caspase-3-like activity exists in xylem cell death. In this study, we showed that caspase-3-like activity is present and is associated with secondary xylem development in Populus tomentosa. The protease responsible for the caspase-3-like activity was purified from poplar secondary xylem using hydrophobic interaction chromatography (HIC), Q anion exchange chromatography and gel filtration chromatography. After identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS), it was revealed that the 20S proteasome (20SP) was responsible for the caspase-3-like activity in secondary xylem development. In poplar 20SP, there are seven α subunits encoded by 12 genes and seven β subunits encoded by 12 genes. Pharmacological assays showed that Ac-DEVD-CHO, a caspase-3 inhibitor, suppressed xylem differentiation in the veins of Arabidopsis cotyledons. Furthermore, clasto-lactacystin β-lactone, a proteasome inhibitor, inhibited PCD of tracheary element in a VND6-induced Arabidopsis xylogenic culture. In conclusion, the 20S proteasome is responsible for caspase-3-like activity and is involved in xylem development.     

Regulation of monocyte apoptosis by the protein kinase Cdelta-dependent phosphorylation of caspase-3

Monocytes are central components of the innate immune response and normally circulate for a short period of time before undergoing spontaneous apoptosis. During inflammation, differentiation, or oncogenic transformation, the life span of monocytes is prolonged by preventing the activation of the apoptotic program. Here we showed that caspase-3, a cysteine protease required for monocyte apoptosis, is a phosphoprotein. We identified protein kinase Cdelta (PKCdelta) as a member of the protein kinase C family that associates with and phosphorylates caspase-3. The PKCdelta-dependent phosphorylation of caspase-3 resulted in an increase in the activity of caspase-3. This effect of PKCdelta is specific to caspase-3, as evidenced by the absence of similar effects on caspase-9. The activity of PKCdelta precedes the activation of caspase-3 during spontaneous monocyte apoptosis and in monocyte-induced apoptosis. We found that the overexpression of PKCdelta resulted in an increase of apoptosis, whereas its inhibition blocked caspase-3 activity and decreased apoptosis. Our results provided evidence that the PKCdelta-dependent phosphorylation of caspase-3 provided a novel pro-apoptotic mechanism involved in the regulation of monocyte life span.     

Regulation of proteasome activity in health and disease

The ubiquitin–proteasome system (UPS) is the primary selective degradation system in the nuclei and cytoplasm of eukaryotic cells, required for the turnover of myriad soluble proteins. The hundreds of factors that comprise the UPS include an enzymatic cascade that tags proteins for degradation via the covalent attachment of a poly-ubiquitin chain, and a large multimeric enzyme that degrades ubiquitinated proteins, the proteasome. Protein degradation by the UPS regulates many pathways and is a crucial component of the cellular proteostasis network. Dysfunction of the ubiquitination machinery or the proteolytic activity of the proteasome is associated with numerous human diseases. In this review we discuss the contributions of the proteasome to human pathology, describe mechanisms that regulate the proteolytic capacity of the proteasome, and discuss strategies to modulate proteasome function as a therapeutic approach to ameliorate diseases associated with altered UPS function. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis

We assessed viable Pax7(-/-) mice in 129Sv/J background and observed reduced growth and marked muscle wasting together with a complete absence of functional satellite cells. Acute injury resulted in an extreme deficit in muscle regeneration. However, a small number of regenerated myofibers were detected, suggesting the presence of residual myogenic cells in Pax7-deficient muscle. Rare Pax3(+)/MyoD+ myoblasts were recovered from Pax7(-/-) muscle homogenates and cultures of myofiber bundles but not from single myofibers free of interstitial tissues. Finally, we identified Pax3+ cells in the muscle interstitial environment and demonstrated that they coexpressed MyoD during regeneration. Sublaminar satellite cells in hind limb muscle did not express detectable levels of Pax3 protein or messenger RNA. Therefore, we conclude that interstitial Pax3+ cells represent a novel myogenic population that is distinct from the sublaminar satellite cell lineage and that Pax7 is essential for the formation of functional myogenic progenitors from sublaminar satellite cells.     

p53 protein and p21 mRNA levels and caspase-3 activity are altered by zinc status in aortic endothelial cells

The influence ofzinc status on the levels of p53, as well as downstream targetsof p53 in cell repair and survival, was examined in human aorticendothelial cells (HAECs). A serum-reduced low-zinc medium (ZD) wasused to deplete zinc over one passage. Other treatments includedzinc-normal control (ZN), zinc-adequate (ZA), and zinc-supplemented (ZS) treatment with 3.0, 16.0, and 32.0 µM zinc, respectively. Cellular zinc levels in the ZD cells were 64% of ZN controls; levelsin the ZA cells were not different, but levels in ZS cells weresignificantly higher (40%) than in ZN cells. No difference in p53 mRNAabundance was detected among all treatments; however, p53 nuclearprotein levels were >100% higher in the ZD and ZS cells and almost200% higher in the ZA cells than in ZN controls. In addition, p21 mRNAabundance, a downstream target of p53 protein, was increased in the ZScells compared with both the ZN control and ZD cells. In the ZS cells,bax and mcl-1 were also ~50% higher compared with ZN controls,whereas bcl-2 mRNA was increased compared with ZA cells. Moreover,caspase-3 activity of ZD cells was not different from that of ZNcontrols but was reduced to 83 and 69% of ZN controls in ZA and ZScells, respectively. Thus p53 protein and p53 downstream target genesappeared to be modulated by intracellular zinc status in HAECs.

     

Regulation of the Apaf-1/caspase-9 apoptosome by caspase-3 and XIAP

The apoptosome is a multiprotein complex comprising Apaf-1, cytochrome c, and caspase-9 that functions to activate caspase-3 downstream of mitochondria in response to apoptotic signals. Binding of cytochrome c and dATP to Apaf-1 in the cytosol leads to the assembly of a heptameric complex in which each Apaf-1 subunit is bound noncovalently to a procaspase-9 subunit via their respective CARD domains. Assembly of the apoptosome results in the proteolytic cleavage of procaspase-9 at the cleavage site PEPD(315) to yield the large (p35) and small (p12) caspase-9 subunits. In addition to the PEPD site, caspase-9 contains a caspase-3 cleavage site (DQLD(330)), which when cleaved, produces a smaller p10 subunit in which the NH(2)-terminal 15 amino acids of p12, including the XIAP BIR3 binding motif, are removed. Using purified proteins in a reconstituted reaction in vitro, we have assessed the relative impact of Asp(315) and Asp(330) cleavage on caspase-9 activity within the apoptosome. In addition, we characterized the effect of caspase-3 feedback cleavage of caspase-9 on the rate of caspase-3 activation, and the potential ramifications of Asp(330) cleavage on XIAP-mediated inhibition of the apoptosome. We have found that cleavage of procaspase-9 at Asp(330) to generate p35, p10 or p37, p10 forms resulted in a significant increase (up to 8-fold) in apoptosome activity compared with p35/p12. The significance of this increase was demonstrated by the near complete loss of apoptosome-mediated caspase-3 activity when a point mutant (D330A) of procaspase-9 was substituted for wild-type procaspase-9 in the apoptosome. In addition, cleavage at Asp(330) exposed a novel p10 NH(2)-terminal peptide motif (AISS) that retained the ability to mediate XIAP inhibition of caspase-9. Thus, whereas feedback cleavage of caspase-9 by caspase-3 significantly increases the activity of the apoptosome, it does little to attenuate its sensitivity to inhibition by XIAP.     

Regulation of proteasome activity in activated human platelets

Ubiquitin-proteasome system has emerged a central player in regulation of diverse cellular processes. However, relevance of proteasome activity in platelets, which are terminally differentiated enucleate cells, is not clear. In this report we show that activation of platelets with physiological agonists was associated with 7-10 -fold rise in proteasomal activity. Elevation of cytosolic calcium with A23187 or thapsigargin resulted in significant increase in enzymatic activity, while treatment with intracellular calcium chelator or inhibitor of inositol trisphosphate receptor attenuated proteasomal enzymes in collagen-stimulated platelets. Specific inhibitors of protein kinase C as well as calpain, too, downregulated proteasome function. To conclude, proteasomal enzymatic activity in platelets is regulated by cytosolic calcium through Ca(2+)-dependent downstream effectors like calpain and protein kinase C.