Insulin-like growth factors (IGFs) are critical for development and growth of skeletal muscles, but because several tissues produce IGFs, it is not clear which source is necessary or sufficient for muscle growth. Because it is critical for production of both IGF-I and IGF-II, we ablated glucose-regulated protein 94 (GRP94) in murine striated muscle to test the necessity of local IGFs for normal muscle growth. These mice exhibited smaller skeletal muscles with diminished IGF contents but with normal contractile function and no apparent endoplasmic reticulum stress response. This result shows that muscles rely on GRP94 primarily to support local production of IGFs, a pool that is necessary for normal muscle growth. In addition, body weights were ∼30% smaller than those of littermate controls, and circulating IGF-I also decreased significantly, yet glucose homeostasis was maintained with little disruption to the growth hormone pathway. The growth defect was complemented on administration of recombinant IGF-I. Thus, unlike liver production of IGF-I, muscle IGF-I is necessary not only locally but also globally for whole-body growth.

Insulin-like growth factor I (IGF-I) coordinates proliferation and differentiation in a wide variety of cell types. The igf1 gene not only produces IGF-I, but also generates multiple carboxy-terminal extensions, the E-peptides, through alternative splicing leading to different isoforms. It is not known if the IGF-I isoforms share a common pathway for their actions, or if there are specific actions of each protein. Viral administration of murine IGF-IA, IGF-IB, and mature IGF, which lacked an E-peptide extension, was utilized to identify IGF-I isoform-specific responsive genes in muscles of young growing mice. Microarray analysis revealed responses that were driven by increased IGF-I regardless of the presence of E-peptide, such as Bcl-XL. In contrast, distinct expression patterns were observed after viral delivery of IGF-IA or IGF-IB, which included matrix metalloproteinase 13 (MMP13). Expression of Bcl-XL was prevented when viral administration of the IGF-I isoforms was performed into muscles of MKR mice, which lack functional IGF-I receptors on the muscle fibers. However, MMP13 expression persisted under the same conditions after viral injection of IGF-IB. At 4 mo after viral delivery, expression of IGF-IA or IGF-IB promoted muscle hypertrophy, but viral delivery of mature IGF-I failed to increase muscle mass. These studies provide evidence that local production of IGF-I requires the E-peptides to drive hypertrophy in growing muscle and that both common and unique pathways exist for the IGF-I isoforms to promote biological effects.

Fetal membranes usually rupture during the process of labor. Premature fetal membrane rupture occurs not infrequently and is associated with significant fetal and maternal morbidity. The mechanisms of normal and pathologic fetal membrane rupture are not well understood. We have examined structural and biochemical changes in the rat amnion as labor approaches in order to characterize this process in normal pregnancy. Here we report that before the onset of active labor the amnion epithelial cells undergo apoptotic cell death which encompasses degradation of 28S ribosomal subunit RNA and associated P proteins and fragmentation of nuclear DNA. Concurrent with these cellular changes, the amnion type I collagen matrix is degraded with the accumulation of three-quarter length type I collagen fragments in extraembryonic fluid, characteristic of the cleavage of fibrillar collagen by interstitial collagenase. Western blot and immunohistochemical analyses confirmed that interstitial collagenase protein appears in association with the loss of amnion type I collagen. We conclude that amnion epithelial cells undergo a process of programmed cell death associated with orchestrated extracellular matrix degradation which begins before the onset of active labor. Thus, fetal membrane rupture is likely to be the result of biochemical changes as well as physical forces. ( J. Clin. Invest. 1996.

HIV protease inhibitors (HIV PI) are a class of antiretroviral drugs that are designed to target the viral protease. Unexpectedly, this class of drugs is also reported to have antitumor activity. In this study, we have evaluated the in vitro activity of nelfinavir, a HIV PI, against human melanoma cells. Nelfinavir inhibits the growth of melanoma cell lines at low micromolar concentrations that are clinically attainable. Nelfinavir promotes apoptosis and arrests cell cycle at G 1 phase. Cell cycle arrest is attributed to inhibition of cyclindependent kinase 2 (CDK2) and concomitant dephosphorylation of retinoblastoma tumor suppressor. We further show that nelfinavir inhibits CDK2 through proteasome-dependent degradation of Cdc25A phosphatase. Our results suggest that nelfinavir is a promising candidate chemotherapeutic agent for advanced melanoma, for which novel and effective therapies are urgently needed. [Cancer Res 2007;67(3):1221-7]

Lei H, Leong D, Smith LR, Barton ER. Matrix metalloproteinase 13 is a new contributor to skeletal muscle regeneration and critical for myoblast migration. Am J Physiol Cell Physiol 305: C529-C538, 2013. First published June 12, 2013 doi:10.1152/ajpcell.00051.2013.-Efficient skeletal muscle repair and regeneration require coordinated remodeling of the extracellular matrix (ECM). Previous reports have indicated that matrix metalloproteinases (MMPs) play the pivotal role in ECM remodeling during muscle regeneration. The goal of the current study was to determine if the interstitial collagenase MMP-13 was involved in the muscle repair process. Using intramuscular cardiotoxin injections to induce acute muscle injury, we found that MMP-13 expression and activity transiently increased during the regeneration process. In addition, in muscles from mdx mice, which exhibit chronic injury, MMP-13 expression and protein levels were elevated. In differentiating C2C12 cells, a murine myoblast cell line, Mmp13 expression was most pronounced after myoblast fusion and during myotube formation. Using pharmacological inhibition of MMP-13 to test whether MMP-13 activity is necessary for the proliferation, differentiation, migration, and fusion of C2C12 cells, we found a dramatic blockade of myoblast migration, as well as a delay in differentiation. In contrast, C2C12 cells with stable overexpression of MMP-13 showed enhanced migration, without affecting myoblast maturation. Taken together, these results support a primary role for MMP-13 in myoblast migration that leads to secondary effects on differentiation.collagenase; matrix metalloproteinase; muscle repair; myoblast maturation

Insulin-like growth factor (IGF)-I is a critical protein for cell development and growth. Alternative splicing of the igf1 gene gives rise to multiple isoforms. In rodents, proIGF-IA and proIGF-IB have different carboxy-terminal extensions called the E-peptides (EA and EB) and upon further posttranslational processing, produce the identical mature IGF-I protein. Rodent EB has been reported to have mitogenic and motogenic effects independent of IGF-I. However, effects of EA or EB on mature IGF-I, or whether proIGF-IA and proIGF-IB have different properties, have not been addressed. To determine whether the presence of EA or EB affected the distribution and stability of mature IGF-I protein, transient transfections of cDNAs encoding murine IGF-IA, IGF-IB, and mature IGF-I were performed in C2C12 cells, a skeletal muscle cell line. IGF-I secretion was measured by enzyme-linked immunosorbent assay of the media, and did not differ between expression of proIGF-IA, proIGF-IB, or mature IGF-I expression. Next, epitope-tagged constructs were transfected to determine cellular distribution of IGF-I, EA, and EB in the cells throughout the culture. IGF-I was detected in significantly fewer nontransfected cells in cultures transfected with mature IGF-I compared with transfection of proIGF-IA or proIGF-IB. These results demonstrate that EA and EB are not required for IGF-I secretion but that they increase cell entry of IGF-I from the media. This study provides evidence that the EA and EB may modulate IGF-I in addition to having independent activity.

The fetal membranes undergo striking changes in structure before delivery that involve catabolism of the extracellular matrix. To investigate the role of specific enzymes in this process, we examined gelatinase activities in rat amnion, visceral yolk sac placenta, and placenta and amniotic fluid between Days 18-21 of pregnancy. Matrix metalloproteinase (MMP)-2 was present in amnion on all days, and its activity increased slightly on Day 21. The 92-kDa gelatinase, MMP-9, was not detected on Days 18-20 but appeared by the morning of Day 21. There was a marked increase in MMP-9 mRNA in the amnion on Day 20, preceding the appearance of MMP-9 activity. Western blotting confirmed an increase in MMP-9 protein in amnion on Day 21. MMP-2 and MMP-9 activities were detected in extracts of whole yolk sac placenta, placenta, and amniotic fluid, but there were no striking changes in these gelatinases between Days 18 and 21. However, the capsular regions of the visceral yolk sac placentae, which thin and rupture during labor, did show higher MMP-9 activity on Day 21 than on Days 18 and 20. We suggest that the striking increase in MMP-9 expression in amnion and possibly the capsular region of the visceral yolk sac placenta approximately 12 h prior to delivery is responsible, in part, for the alterations in the structure of these fetal membranes before parturition.

Insulin-like growth factors (IGFs) are essential for local skeletal muscle growth and organismal physiology, but these actions are entwined with glucose homeostasis through convergence with insulin signaling. The objective of this work was to determine whether the effects of IGF-I on growth and metabolism could be separated. We generated muscle-specific IGF-I-deficient (MID) mice that afford inducible deletion of Igf1 at any age. After Igf1 deletion at birth or in young adult mice, evaluations of muscle physiology and glucose homeostasis were performed up to 16 wk of age. MID mice generated at birth had lower muscle and circulating IGF-I, decreased muscle and body mass, and impaired muscle force production. Eight-wk-old male MID had heightened insulin levels with trends of elevated fasting glucose. This phenotype progressed to impaired glucose handling and increased fat deposition without significant muscle mass loss at 16 wk of age. The same phenotype emerged in 16-wk-old MID mice induced at 12 wk of age, compounded with heightened muscle fatigability and exercise intolerance. We assert that muscle IGF-I independently modulates anabolism and metabolism in an age-dependent manner, thus positioning muscle IGF-I maintenance to be critical for both muscle growth and metabolic homeostasis.-Vassilakos, G., Lei, H., Yang, Y., Puglise, J., Matheny, M., Durzynska, J., Ozery, M., Bennett, K., Spradlin, R., Bonanno, H., Park, S., Ahima, R. S., Barton, E. R. Deletion of muscle Igf-I transiently impairs growth and progressively disrupts glucose homeostasis in male mice.