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J R Florini

Publications and source records attributed to J R Florini.

At least 37 records · Page 2Linked to original sources
PubMed1990

Highly specific inhibition of IGF-I-stimulated differentiation by an antisense oligodeoxyribonucleotide to myogenin mRNA. No effects on other actions of IGF-T.

Myogenin is a member of the recently discovered family of muscle determination genes that have been shown to induce myogenic differentiation in nonmuscle cells and to be closely correlated with terminal differentiation in myoblasts. An antisense oligodeoxyribonucleotide complementary to the first five codons of myogenin blocks the stimulation of terminal myogenic differentiation by insulin-like growth factor I (IGF-I). This effect exhibits a high degree of specificity on two levels; exchanging the positions as few as 2 of the 15 bases in the oligomer abolishes its activity, and none of the other processes stimulated by IGF-I in L6A1 myoblasts are affected by the presence of the oligomer. These processes include cell proliferation as well as incorporation of leucine, uridine, and thymidine into macromolecules. The specificity, ease, and convenience of this approach indicates its potential applicability to studies on actions of other putative controlling genes in other systems.

Animals↗
PubMed1990

Effect of age and IGF-I administration on elastin gene expression in rat aorta.

An age-related decrease in elasticity of arteries has been found in clinical and experimental studies done during the past two decades. We have investigated molecular and endocrine aspects of that decrease by examining the effects of age and insulin-like growth factor-I (IGF-I) on rat aorta elastogenesis. For comparison, pulmonary elastogenesis was examined in the same experimental animals. Different aged groups of male Fischer 344 rats (barrier protected) were implanted with minipumps for a two-week infusion of either 0.1 N acetic acid (vehicle solution) or IGF-I (1.2 mg/kg/day). The DNA content (micrograms DNA/g tissue) decreased with age in aorta but remained fairly constant in lung. Administration of IGF-I increased the aortic DNA content in all but the oldest rats. Conversely, the DNA content of pulmonary tissue was significantly increased in only the youngest animals. The steady-state levels of tropoelastin mRNA decreased dramatically in both aorta and lung with increased age. The decrease was greater in lung than aorta. Administration of IGF-I elevated aortic tropoelastin mRNA steady-state levels, whereas lung tropoelastin mRNA levels were unaffected by IGF-I administration. Aortic tissue synthesized decreased amounts of insoluble elastin with increased age. These results establish a direct relationship between aortic tropoelastin mRNA levels and the synthesis of insoluble elastin in aging. Administration of IGF-I increased aortic elastin synthesis throughout the life span of the rat, although the proportionate increase diminished with age.

Aging↗
PubMed1989

Assay of creatine kinase in microtiter plates using thio-NAD to allow monitoring at 405 nM.

An assay system for creatine kinase using microtiter plates and a plate reader that records absorbancies at 405 nM has been devised. The system is an adaptation of well-established assays that couple creatine kinase with the reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase (G6PDH), to give a measurable increase in reduced pyridine nucleotide quantitated by absorbance at 340 nM. Two features of this system are modified for reading at 405 nM: (i) The thioamido derivative of NAD is used because its reduced form exhibits a substantial increase in absorbance at 405 nM, the most commonly available wavelength on microplate readers; and (ii) glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is used because it can reduce either NAD or NADP (unlike most other G6PDH enzymes, which require NADP), thus making it unnecessary to use the more expensive thio-NADP. The rate of thio-NAD reduction is linear with enzyme concentration and time over a 20-fold range of concentrations of purified creatine kinase, and the assay also works well with myogenic cells allowed to grow and differentiate in the 96-well plate in which the assay is performed. This system offers considerable savings in cells, time, and material in studies of muscle cell differentiation, for which creatine kinase levels are frequently measured. It also provides a potential method for the convenient and economical measurement of activities of many other enzymes that can be coupled to reduction of thio-NAD.

Animals↗
PubMed1989

Limitations of interpretation of age-related changes in hormone levels: illustration by effects of thyroid hormones on cardiac and skeletal muscle.

Studies on age-related changes in hormone secretion or circulation are frequently interpreted to indicate changes in the functions of target tissues for that hormone. Such interpretations are not necessarily correct. For instance, the levels of thyroid hormone change modestly with age, and responses to this change differ dramatically in cardiac and skeletal muscles. There is a substantial change in myosin isozyme content of the heart, but little or no change in skeletal muscle fiber type distribution in specific pathogen free (SPF) barrier-protected rats. These changes (or lack of them) are reflected in functional differences (or lack of them) in the target tissues. The most obvious difference between the recent studies showing no differences with age in skeletal muscle fiber distribution and previous reports of general decreases with age in content of fast fibers is in animal housing conditions; all of the animals showing an age-related change had been housed in conventional colonies, while those exhibiting little or no change were in SPF barrier-protected facilities.

Aging↗
PubMed1989

Skeletal muscle fiber types and myosin ATPase activity do not change with age or growth hormone administration.

Fischer 344 rats at various ages throughout the life span have been treated with growth hormone, clonidine, and insulin-like growth factor-I to restore circulating somatomedin levels in old animals to levels found in younger rats. The injections were intended to reverse (2-week treatments) or prevent (6-month treatments) deleterious effects of aging on skeletal muscle--specifically the loss of fast fibers during the latter part of the life span. However, measurements of myosin ATPase (and subsequent histochemical fiber type determinations) revealed that the previously reported age-related decrease in ATPase activities and fast fiber content did not occur in barrier-protected specific pathogen free rats or mice. None of the treatments used had a significant effect on the ATPase activity or fiber composition of soleus, extensor digitorum longus, or diaphragm muscles, although parallel determinations in collaborating laboratories verified that the hormones had major effects in other systems. Previously reported decreases with age in ATPase activity of heart muscle were confirmed in our experiments, and these decreases were shown to reflect a change in myosin isozyme composition of the hearts, both at the protein and the mRNA levels of gene expression.

Aging↗
PubMed1989

Effects of growth factors on myogenic differentiation.

It has now been well established that the terminal differentiation of muscle cells in culture is subject to control by hormones and growth factors in the incubation medium. Thus far the most potent and most extensively studied agents are fibroblast growth factor (FGF), the insulinlike growth factors (IGFs), and transforming growth factor-beta (TGF-beta). Independent reports from several laboratories have established that both FGF and TGF-beta are potent inhibitors of differentiation and both appear to act at early stages of commitment to differentiation. Stimulation of differentiation by the IGFs (and by insulin at concentrations in the microgram/ml range) has also been observed and confirmed repeatedly. FGF and IGF are mitogenic for muscle cells, and TGF-beta either has no effect or suppresses cell proliferation, so previous generalizations that mitogens inhibit myogenic differentiation are clearly not valid when results with purified agents in well-defined media are considered. Work with oncogenes and specific toxins is beginning to reveal the mechanisms by which these agents might affect differentiation, and there is reason for optimism that an understanding of the molecular events that control terminal differentiation may be attained in the near future.

Animals↗
PubMed1988

Decrease in transforming growth factor-beta binding and action during differentiation in muscle cells.

We report here the effects of differentiation on the binding and action of transforming growth factor-beta (TGF-beta) in three lines of myogenic cells. In two lines (L6-A1 and C2) which irreversibly differentiate by fusing to form postmitotic myotubes, there is a virtual disappearance of TGF-beta binding sites as differentiation occurs. Analyses of the binding curves by the method of Scatchard indicates that there is little or no change in affinity but a substantial decrease in the number of binding sites. In L6-A1 cells, responsiveness to TGF-beta decreases in parallel to the loss of receptors. The decreases in TGF-beta binding and activity with differentiation are not paralleled by similar changes in another growth factor, insulin-like growth factor-I, which exhibits little change in binding and only a modest decrease in activity as L6-A1 myoblasts differentiate to form myotubes. In a third cell line (BC3H1), which exhibits reversible differentiation without fusion, there is little or no change in TGF-beta binding as the cells differentiate. Comparisons with reported decreases in binding of fibroblast and epidermal growth factors indicates that there are substantial differences in growth factor binding and actions as muscle cells differentiate, but it is not possible to make the simple generalization that differentiation is accompanied by a decrease in binding of all growth factors.

Animals↗
PubMed1988

Actions of transforming growth factor-beta on muscle cells.

It has recently been reported by three laboratories that transforming growth factor-beta (TGF-beta) is a potent and reversible inhibitor of differentiation in myogenic cells. To improve our understanding of this inhibition, we investigated the effects of TGF-beta on several other processes in L6 myoblasts, with emphasis on actions of the insulin-like hormones (which stimulate myoblast differentiation). We found that TGF-beta had no effect on the binding of insulin-like growth factors (IGFs) to their receptors on the cell surface, and it had little or no effect on some actions of the IGFs. There was essentially no change in the suppression of proteolysis or the stimulation of cell proliferation by IGFs when TGF-beta was also added to the medium. However, there was an effect of TGF-beta on another process stimulated by the IGFs; TGF-beta was an equally active and more potent stimulator of amino acid uptake than was IGF-I, and the stimulation was additive beyond the maximal response attained with IGF-I, suggesting that the two act by different mechanisms. TGF-beta had significant effects on myoblast morphology, causing the formation of abundant stress fibers containing cytoplasmic (but not myofibrillar) actin. Addition of TGF-beta at various times after initiation of differentiation demonstrated that TGF-beta inhibits an early process in differentiation. Thus it appears that the interactions of TGF-beta and the IGFs in myoblasts are complex; in some instances the effects of IGFs are inhibited and in others they are mimicked or are unaffected. It is clear that TGF-beta does not act by simply interfering with IGF binding or blocking early steps in its action on myoblasts.

Amino Acids↗
PubMed1987

Hormonal control of muscle growth.

In muscle of whole animals, pituitary growth hormone, the thyroid hormones, and insulin are major growth-promoting hormones, and the glucocorticoids have significant catabolic actions. At the cellular level the primary anabolic hormones for cultured myoblasts are the somatomedins (insulin-like growth factors) and fibroblast growth factor. In these cells physiological concentrations of growth hormone, thyroid hormones, and insulin have no growth-promoting effect; some of the reported actions of insulin probably result from cross-reaction with the somatomedin receptor. Results with purified proteins do not support the view that mitogens block myoblast differentiation; transforming growth factor-beta and interferon are nonmitogenic proteins that inhibit differentiation, insulin-like growth factors are mitogens that stimulate differentiation, and fibroblast growth factor is the only purified mitogen that inhibits differentiation. At least six serum-free media have now been devised for the growth of various kinds of muscle cells under closely defined conditions.

Animals↗
PubMed1987

Insulin-like growth factor I, somatomedin C, induces the synthesis of tropoelastin in aortic tissue.

The action of IGF-I (insulin-like growth factor I) on the synthesis of tropoelastin in chick embryonic aortae was examined. Maximal and selective stimulation of the relative (40%) and absolute (145%) rates of tropoelastin synthesis over control occurred at an IGF-I concentration of 100 ng/ml of medium. Parallel to the increase in synthesis was a 92% increase in the amount of tropoelastin activity per 100 ng of poly (A)+ RNA translated in a cell-free system. The relative rate of tropoelastin synthesis achieved at maximal stimulation is greater than that observed during normal aortic embryogenesis. The stimulatory action of the hormone on elastin synthesis appears to be at a pretransitional level perhaps involving increased transcription or stabilization of the tropoelastin mRNAs. These results suggest that IGF-I may play a key role in the regulation of elastogenesis in arterial tissue.

Animals↗
PubMed1987

The type II insulin-like growth factor (IGF) receptor has low affinity for IGF-I analogs: pleiotypic actions of IGFs on myoblasts are apparently mediated by the type I receptor.

We have characterized binding and several actions of the somatomedins [insulin-like growth factors I and II (IGF-I and IGF-II)] on L6 myoblasts. Both IGF-I and IGF-II are potent stimulators of amino acid uptake, cell proliferation, and differentiation; they also suppress protein degradation in these cells. In all measurements, the relative potencies are IGF-I greater than IGF-II greater than insulin. Two recombinant DNA-produced analogs of IGF-I, (Thr59)IGF-I and (N-Met)IGF-I, were as active as native IGF-I in all four assays. However, when 125I-labeled hormones were used for studies of binding to IGF receptors, there was a striking difference between the native and recombinant IGF-I molecules. Both were bound significantly by the type I receptor (a 350K molecule that is dissociated upon sulfhydryl reduction), but the recombinant analogs exhibited little cross-reactivity with the type II receptor (a 220K molecule that is not dissociated by reduction). Thus, the equal activity of native IGF-I and its recombinant DNA-produced analogs coupled with the higher potency of IGF-I (compared to IGF-II) suggest that the type II receptor plays little or no role in the four actions of the somatomedins studied in L6 myoblasts.

Affinity Labels↗
PubMed1986

Transforming growth factor-beta. A very potent inhibitor of myoblast differentiation, identical to the differentiation inhibitor secreted by Buffalo rat liver cells.

Transforming growth factor-beta (TGF-beta) is now known to have a number of actions in addition to the induction of phenotypic transformation in fibroblastic cells. In this paper, we characterize its inhibition of differentiation in rat myoblasts of Yaffe's L6 strain and demonstrate its identity or very close similarity to the differentiation inhibitor (DI) secreted by Buffalo rat liver cells cultured in serum-free medium. At concentrations as low as 60 pg/ml, TGF-beta gave detectable inhibition of differentiation measured as myoblast fusion and creatine kinase elevation; maximal inhibition was observed at and above 0.5 ng/ml (20 pM). The inhibition persisted as long as fresh TGF-beta was added at 48-h intervals, but it was reversed upon removal of the factor. By itself or in the presence of mitogens, TGF-beta had no mitogenic activity in the L6 cells. Concentration dependencies of human TGF-beta and the rat DI were closely parallel in three assays: inhibition of myoblast differentiation, stimulation of normal rat kidney cell growth in soft agar, and competition for displacement of labeled TGF-beta from binding sites on A549 human lung carcinoma cells. We conclude that most if not all of the DI activity found in medium conditioned by Buffalo rat liver cells can be attributed to the presence of TGF-beta or a very similar molecule. These observations also offer a potentially useful approach to study the control of myogenesis; the process(es) can be blocked in cloned L6 myoblasts by incubation with very small quantities of a pure protein in fully defined serum-free medium.

Animals↗
PubMed1986

Biphasic concentration dependency of stimulation of myoblast differentiation by somatomedins.

It is widely believed that mitogens inhibit in vitro differentiation of myoblasts to form postmitotic myotubes, but we and others have shown that the mitogenic hormones insulin and the insulin-like growth factors (IGFs) stimulate myoblast differentiation. We now report the results of concentration-dependency studies that resolve this disagreement. We found that the IGFs give a biphasic dose-response curve; at low concentrations, there is progressive stimulation of L6 myoblast differentiation; at higher concentrations, there is a progressive decrease. Similar results were obtained with IGF-II and insulin. When differentiation was maximally stimulated (by 1,280 ng/ml insulin), adding rat IGF-II gave decreases in differentiation similar to those reported for other mitogens. Two trivial explanations have been eliminated: stimulation of differentiation (at low concentrations) is not due to enhanced survival or growth of the cells, and inhibition (at higher concentrations) is not a toxic effect. In L6 cells, epidermal growth factor and fibroblast growth factor had no effect on proliferation or differentiation. We conclude that the effects of medium components on myoblast differentiation cannot be generalized to indicate inhibition by all mitogens; depending on the cell lines and concentrations used, certain mitogens may either stimulate or inhibit differentiation.

Animals↗
PubMed1985

Somatomedin-C levels in healthy young and old men: relationship to peak and 24-hour integrated levels of growth hormone.

We investigated the relationship between growth hormone secretion and somatomedin levels as a function of age in normal healthy men. There was a substantial decrease with age in somatomedin levels from 0.95 +/- 0.06 (M +/- SEM) units/ml in young (23 to 27 years) men to 0.68 +/- 0.07 in old (58 to 82 years) men. The blood samples were taken throughout a 24-hour period, so it was possible to analyze the data for correlations between average somatomedin levels and various aspects of growth hormone secretion. There was a significant correlation of somatomedin level with the 24-hour integrated growth hormone level, and a nearly significant correlation with the sum of the highest three peaks, but no correlation with the highest nighttime peak or the basal level of growth hormone. These lower blood levels of somatomedin in the aged may be responsible, at least in part, for the catabolic effects on muscle and bone frequently associated with aging.

Adult↗
PubMed1985

Hormonal regulation of somatomedin secretion by fetal rat hepatocytes in primary culture.

To determine which hormones might regulate somatomedin secretion in the fetus, we measured somatomedin levels in conditioned medium from primary cultures of fetal rat hepatocytes. We employed a bioassay [( 3H]thymidine incorporation into DNA of chick embryo fibroblasts), a displacement assay [competition for binding of radiolabeled multiplication-stimulating activity (rat insulin-like growth factor II) to the somatomedin-binding protein] for total somatomedin, and the RIA for somatomedin-C. Epidermal growth factor and dexamethasone were the most active hormones tested; total somatomedin levels were 2-3 times above control levels. Rat GH was much less stimulatory. Human placental lactogen, glucagon, and insulin had little or no effect. Stimulation of somatomedin secretion by both epidermal growth factor and dexamethasone was time and dose dependent. The maximal response occurred at 48 h at a concentration of about 1 X 10(-7) M of either hormone. In the bioassay, stimulation by epidermal growth factor, but not dexamethasone, was detected. The steroid enhanced the secretion of an inhibitor that completely masked the mitogenic activity of the increased somatomedin levels. The somatomedin secreted by fetal hepatocytes exhibited immunological cross-reactivity with human somatomedin-C, but the levels were 500-fold less than those measured by our displacement assay. This suggests that the predominant fetal rat somatomedin is not somatomedin-C. We conclude that epidermal growth factor and dexamethasone, but not GH or placental lactogen, stimulated the secretion by fetal hepatocytes of a somatomedin which resembled multiplication-stimulating activity.

Animals↗
PubMed1984

The role of polyamines in somatomedin-stimulated differentiation of L6 myoblasts.

The somatomedins are potent stimulators of proliferation and differentiation of cultured myoblasts. In studies on the mechanism(s) of these actions, we have measured the activities of ornithine decarboxylase (ODC), an enzyme associated with rapid cell proliferation, and creatine kinase (CK), a biochemical marker for muscle differentiation, after treatment of L6 myoblast cultures with Multiplication Stimulating Activity (MSA), a member of the somatomedin family of insulinlike growth factors. ODC levels reached a peak 24 hours after MSA addition (before any detectable differentiation of the myoblasts) and then decreased as differentiation commenced and CK activity increased. Addition of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, caused a dramatic decrease in differentiation. Measurement of 3H-thymidine incorporation, DNA content, and cell number established that the effect of DFMO on differentiation was not a simple consequence of its antiproliferative actions. Cellular levels of putrescine and spermidine (but not spermine) decreased substantially following addition of DFMO to the cultures. The inhibitory effects of DFMO were abolished upon addition of exogenous polyamines to the medium. However, addition of polyamines in the absence of MSA or DFMO did not mimic the stimulation of differentiation by MSA. We conclude that polyamines play an essential role in the stimulation of L6 myoblast differentiation by somatomedins, but they are not sufficient to effect this stimulation.

Animals↗
PubMed1984

Stimulation and inhibition of myoblast differentiation by hormones.

The growth and differentiation of L6 myoblasts are subject to control by two proteins secreted by cells of the Buffalo rat liver line. The first of these, rat insulinlike growth factor-II (formerly designated multiplication stimulating activity) is a potent stimulator of myoblast proliferation and differentiation, as well as associated processes such as amino acid uptake and incorporation into protein, RNA synthesis, and thymidine incorporation into DNA. In addition, this hormone causes a significant decrease in the rate of protein degradation. All of these actions seem to be attributable to a single molecular species, although their time courses and sensitivity to the hormone differ substantially. The second protein, the differentiation inhibitor (DI), is a nonmitogenic inhibitor of all tested aspects of myoblast differentiation, including fusion and the elevation of creatine kinase. Indirect immunofluorescence experiments demonstrated that DI also blocks accumulation of myosin heavy chain and myomesin. Upon removal of DI after 72 h incubation, all of these effects were reversed and normal myotubes containing the usual complement of muscle-specific proteins were formed. Thus, this system makes it possible to achieve specific stimulation or inhibition of muscle cell differentiation by addition of purified proteins to cloned cells in serum-free medium.

Animals↗