Brett Langley

Titin-cap associates with, and regulates secretion of, Myostatin

Myostatin, a secreted growth factor, is a key negative regulator of skeletal muscle growth. To identify modifiers of Myostatin function, we screened for Myostatin interacting proteins. Using a yeast two‐hybrid screen, we identified Titin‐cap (T‐cap) protein as interacting with Myostatin. T‐cap is a sarcomeric protein that binds to the N‐terminal domain of Titin and is a substrate of the titin kinase. Mammalian two‐hybrid studies, in vitro binding assays and protein truncations in the yeast two‐hybrid system verified the specific interaction between processed mature Myostatin and full‐length T‐cap. Analysis of protein–protein interaction using surface plasmon resonance (Biacore, Uppsala, Sweden) kinetics revealed a high affinity between Myostatin and T‐cap with a KD of 40 nM. When T‐cap was stably overexpressed in C2C12 myoblasts, the rate of cell proliferation was significantly increased. Western analyses showed that production and processing of Myostatin were not altered in cells overexpressing T‐cap, but an increase in the retention of mature Myostatin indicated that T‐cap may block Myostatin secretion. Bioassay for Myostatin confirmed that conditioned media from myoblasts overexpressing T‐cap contained lower levels of Myostatin. Given that Myostatin negatively regulates myoblast proliferation, the increase in proliferation observed in myoblasts overexpressing T‐cap could thus be due to reduced Myostatin secretion. These results suggest that T‐cap, by interacting with Myostatin, controls Myostatin secretion in myogenic precursor cells without affecting the processing step of precursor Myostatin. J. Cell. Physiol. 193: 120–131, 2002.

Myostatin in muscle growth and repair.

SHARMA, M., B. LANGLEY, J. BASS, and R. KAMBADUR. Myostatin in muscle growth and repair. Exerc. Sport Sci. Rev., Vol. 29, No. 4, pp 155–158, 2001. Myostatin, a member of the TGF beta superfamily, regulates skeletal muscle size by controlling embryonic myoblast proliferation. Recent results show that myostatin may also have a role in muscle regeneration and muscle wasting of adult animals. This review summarizes the recent developments in the regulation of myostatin gene expression and mechanism of its function.

Myostatin inhibits rhabdomyosarcoma cell proliferation through an Rb-independent pathway.

Rhabdomyosarcoma (RMS) tumors are the most common soft-tissue sarcomas in childhood. In this investigation, we show that myostatin, a skeletal muscle-specific inhibitor of growth and differentiation is expressed and translated in the cultured RMS cell line, RD. The addition of exogenous recombinant myostatin inhibits the proliferation of RD cells cultured in growth media, consistent with the role of myostatin in normal myoblast proliferation inhibition. However, unlike normal myoblasts, upregulation of p21 was not observed. Rather, myostatin signalling resulted in the specific downregulation of both Cdk2 and its cognate partner, cyclin-E. The analysis of Rb reveals that there was no change in its phosphorylation status with myostatin treatment, consistent with D-type-cyclin–Cdk4/6 complexes being active in the absence of p21. Moreover, the activity of Rb appeared to be unchanged between treated and nontreated RD cells, as determined by the ability of Rb to bind E2F1. The examination of NPAT, a substrate of cyclin-E–Cdk2 involved in the transcriptional activation of replication-dependent histone gene expression, revealed that it undergoes a loss of phosphorylation with myostatin treatment. Supporting this, a downregulation in H4-histone gene expression was observed. These results suggest that myostatin could potentially be used as an inhibitor of RMS proliferation and define a previously uncharacterized, Rb-independent mechanism for the inhibition of muscle precursor cell proliferation by myostatin.

Milk whey protein concentration and mRNA associated with β-lactoglobulin phenotype

Two common genetic variants of β-lactoglobulin (β-lg), A and B, exist as co- dominant alleles in dairy cattle (Aschaffenburg, 1968). Numerous studies have shown that cows homozygous for β-lg A have more β-lg and less α-lactalbumin (α-la) and casein in their milk than cows expressing only the B variant of β-lg (Ng-Kwai-Hang et al . 1987; Graml et al . 1989; Hill, 1993; Hill et al . 1995, 1997). These differences have a significant impact on the processing characteristics of the milk. For instance, the moisture-adjusted yield of Cheddar cheese is up to 10% higher using milk from cows of the β-lg BB phenotype compared with milk from cows expressing only the A variant (Hill et al . 1997). All these studies, however, describe compositional differences associated with β-lg phenotype in established lactation only. No information is available on the first few weeks of lactation, when there are marked changes in the concentrations of β-lg and α-la (Perez et al . 1990).

Introduction of a Proximal Stat5 Site in the Murine α-Lactalbumin Promoter Induces Prolactin Dependency In Vitro and Improves Expression Frequency In Vivo

In order to establish a possible correlation between in vitro prolactin induction and the transcriptional activity of mammary gene promoters in transgenic mice, a functional Stat5-binding site was created by means of site-directed mutagenesis at position −70 on a 560 bp murine α-lactalbumin promotor linked to a CAT reporter gene. Surprisingly, the wild-type promoter was constitutively active in vitro and could not be induced by prolactin. Introducing the proximal Stat5 site abolished this constitutive activity and resulted in prolactin dependence in both CHO-K1- and HC11-transfected cells. In transgenic mice, both the frequency of lines expressing the transgene and the prevalence of mid to late pregnancy expression were increased.

Rescue of an MMTV transgene by co-integration reveals novel locus control properties of the ovine beta-lactoglobulin gene that confer locus commitment to heterogeneous tissues.

In an attempt to enhance the frequency and level of expression of a poor-performing MMTV-driven transgene, we co-integrated this construct with the ovine β-lactoglobulin (BLG) gene in transgenic mice. Seven lines of transgenic mice possessing co-integrated BLG and MMTV-RZ5 transgenes were compared with 12 lines of mice that possessed only the MMTV-RZ5 construct. Co-integration enhanced the frequency of expression in the mammary gland from two out of 12 lines for the MMTV-RZ5 transgene alone, to five out of seven when co-integrated with BLG. Surprisingly, co-integration also resulted in co-expression of the two transgenes in the salivary gland, lung and spleen in addition to the mammary gland. Furthermore, both transgenes were expressed in virgin animals, and throughout pregnancy and lactation, suggesting that the developmental regulation of the locus follows that of the MMTV-promoter. These findings represent a novel locus control property of the ovine BLG gene that confer s commitment of the locus to the mammary gland, but also to a range of heterogeneous tissues possibly defined by the second promoter at the locus