A role for protein kinase C activity in interleukin-1 (IL-1) induction of IL-2 gene expression but not in IL-1 signal transduction.

Interleukin-1 (IL-1) is known to synergize with phorbol esters in the induction of interleukin-2 (IL-2) expression in T-lymphoid leukemia cells and proliferation of mouse thymocytes. We used a plasmid construct containing the bacterial gene for chloramphenicol acetyltransferase under the control of the human IL-2 promoter to study the nature of this synergism in the murine thymoma cell line EL4. Although IL-1 induction of the IL-2 promoter in these cells required costimulus with phorbol myristate acetate, the signal induced by IL-1 was qualitatively different. We provide evidence to support the hypothesis that the phorbol ester signal is mediated by protein kinase C, and we show that the IL-1 signal is not. That IL-1 and phorbol myristate acetate represent different stimuli was shown by their response to protein kinase C inhibitors, capacity to synergize with increased intracellular free calcium, and requirement for protein synthesis. In addition we show that pretreatment with IL-1 can prime EL4 cells to subsequent activation by concentrations of phorbol esters not normally sufficient to induce IL-2 expression. Pretreated cells remained primed for at least 40 h after removal of the IL-1. Neither phorbol myristate acetate nor a calcium ionophore was capable of preactivating EL4 cells.     

The AP-1 sequence is necessary but not sufficient for phorbol induction of collagenase in fibroblasts.

Collagenase, the only enzyme active at neutral pH that initiates collagen degradation, is a major gene product of fibroblasts that have been stimulated with a variety of agents, including phorbol esters. To study mechanisms controlling collagenase gene expression, we transiently transfected rabbit synovial fibroblasts with chimeric constructs containing up to 1.2 kb of the rabbit collagenase 5'-flanking DNA linked to the chloramphenicol acetyltransferase gene (CAT). Our data indicate that the magnitude of the phorbol response is directly linked to the size of the promoter fragment and that the smallest piece of promoter DNA conferring phorbol inducibility is 127 bp. Deletional and mutational analysis of this fragment revealed that the AP-1 sequence alone is insufficient for phorbol inducibility and the presence of at least two additional sequences (a PEA3-like element and a sequence that includes 5'-TTCA-3') is required. In addition, a substantial increase in responsiveness is seen when a fragment containing 182 bp of 5'-flanking DNA is transfected, implicating a 36 bp region located between -182 and -149 as an enhancer. We conclude (1) that the AP-1 sequence is necessary but insufficient for expression of collagenase in adult fibroblasts, (2) that phorbol inducibility depends on cooperation among several sequence elements within the collagenase promoter, and (3) that regulation of this promoter is more complex than previously described.     

Effect of the phorbol ester TPA on PTH secretion. Evidence for a role for protein kinase C in the control of PTH release

Parathyroid hormone (PTH) secretion is stimulated by low extracellular calcium (Ca2+) in association with a reduction in cyosolic Ca2+, indicating that this cell type does not conform to classical models of stimulus-secretion coupling. We used the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate), which directly activates protein kinase C, to investigate the possible role of this enzyme in the unusual secretory properties of the parathyroid cell. TPA causes a dose-dependent stimulation of PTH release inhibited by high extracellular Ca2+ (EC50 = 10 nM) but has relatively little effect on secretion stimulated by low Ca2+. This effect was mimicked by the beta 4-isomer of phorbol 12,13-didecanoate which also activates kinase C, but not by the alpha 4-isomer, which has no effect on this enzyme. TPA does not modify cellular cAMP or cytosolic Ca2+ in the parathyroid cell indicating that its effects on PTH secretion are not mediated indirectly via changes in these second messengers. These results suggest that inhibition of PTH release at high Ca2+ might be related to a reduction in protein kinase C activity which can be overcome when the enzyme is directly activated by TPA.     

Deletion analysis of the mouse alpha 1(III) collagen promoter.

A chimeric gene was constructed by fusing the DNA sequences containing the 5' flanking region of the mouse alpha 1(III) collagen gene to the coding sequence of the bacterial chloramphenicol acetyltransferase (CAT) gene. Transient transfection experiments indicated that the alpha 1(III) promoter is active in NIH 3T3 fibroblasts and BC3H1 smooth muscle cells. The activity of the alpha 1(III) collagen promoter-CAT plasmid is stimulated approximately ten fold by the presence of the SV40 enhancer element. Removing sequences upstream of -200 stimulates the activity of the chimeric gene eight fold. Further deletion analysis identified sequences located between -350 and -300 that were instrumental in repressing the activity of the promoter. This 50 bp region contains a direct repeat sequence that may be involved in the regulation of the mouse alpha 1(III) collagen gene. Truncating the alpha 1(III) promoter to -80 further stimulated expression. We propose that the positive regulatory elements of this gene appear to be located within the first 80 bp of the promoter, whereas elements located further upstream exert a negative effect on the expression of the gene. Regulation of the alpha 1(III) gene contrasts with that of the alpha 2(I) collagen gene, which appears to be regulated by several positive elements located in various regions of the promoter.     

Role of protein kinase C (PKC) in bone resorption: effect of the specific PKC inhibitor 1-alkyl-2-methylglycerol.

The specific inhibitor of protein kinase C, 1-O-alkyl-2-O-methylglycerol (AMG), was studied for its effect on bone resorption, measured as 45Ca-release, in fetal mouse calvariae. AMG (1 to 50 microM) had no effect on basal bone resorption. AMG inhibited parathyroid hormone (40 nM) induced bone resorption in a dose-dependent manner. Resorption induced by 1,25 (OH)2-vitamin D3 (10 nM) or prostaglandin E2 (5 microM) was also inhibited by AMG. The release of beta-glucuronidase activity paralleled the course of the 45Ca-release. The production of interleukin 6, induced by parathyroid hormone, in fetal rat calvarial osteoblasts was not affected by AMG. AMG (1 to 50 microM) had no cytotoxic effects on cells or calvariae. From these results it is concluded that protein kinase C may have an important role in the regulation of bone resorption.     

Binding of parathyroid hormone to bovine kidney-cortex plasma membranes

1. Plasma membranes were purified from bovine kidney cortex, with a fourfold increase in specific activity of parathyroid hormone-sensitive adenylate cyclase over that in the crude homogenate. The membranes were characterized by enzyme studies. 2. Parathyroid hormone was labelled with (125)I by an enzymic method and the labelled hormone shown to bind to the plasma membranes and to be specifically displaced by unlabelled hormone. Parathyroid hormone labelled by the chloramine-t procedure showed no specific binding. (75)Se-labelled human parathyroid hormone, prepared in cell culture, also bound to the membranes. 3. Parathyroid hormone was shown to retain biological activity after iodination by the enzymic method, but no detectable activity remained after chloramine-t treatment. 4. High concentration of pig insulin inhibited binding of labelled parathyroid hormone to plasma membranes and partially inhibited the hormone-sensitive adenylate cyclase activity in a crude kidney-cortex preparation. 5. EDTA enhanced and Ca(2+) inhibited binding of labelled parathyroid hormone to plasma membranes. 6. Whereas rat kidney homogenates were capable of degrading labelled parathyroid hormone to trichloroacetic acid-soluble fragments, neither crude homogenates nor purified membranes from bovine kidney showed this property. 7. Binding of parathyroid hormone is discussed in relation to metabolism and initial events in hormone action.     

Studies of hormonal regulation of osteocalcin synthesis in cultured fetal rat calvariae

The synthesis of osteocalcin, the major non-collagenous protein of adult bone, was examined in cultures of 21-day fetal rat calvariae. Osteocalcin was measured by a sensitive and specific radioimmunoassay. Osteocalcin concentration in unincubated calvariae was 14.5 +/- 0.5 ng/calvaria. After incubation, there was a continuous increase in bone and medium osteocalcin, and by 96 h the values were about 100% higher than in unincubated calvariae. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) at 10(-11) to 10(-8)M increased osteocalcin synthesis. The effect appeared as early as 6 h after treatment and was primarily observed in the culture medium, and 1,25-(OH)2D3 stimulated osteocalcin up to 9-fold by 96 h. Concomitant with the effect on osteocalcin synthesis, 1,25-(OH)2D3 inhibited collagen synthesis. Cycloheximide markedly decreased osteocalcin concentrations in control and 1,25-(OH)2D3-treated calvariae. The stimulatory effect on osteocalcin synthesis was specific to 1,25-(OH)2D3 since 24,25-dihydroxyvitamin D3, parathyroid hormone, epidermal growth factor, and prostaglandin E2 did not stimulate osteocalcin synthesis, and parathyroid hormone and epidermal growth factor opposed the 1,25-(OH)2D3 stimulatory effect. Insulin did not alter osteocalcin concentration by itself but enhanced the effect of 1,25-(OH)2D3. In conclusion, 1,25-(OH)2D3 stimulates osteocalcin synthesis in cultures of normal calvariae, but this effect is not shared by other hormones known to affect bone metabolism.