Biomedical subjects

D D Bikle

Publications and source records attributed to D D Bikle.

At least 19 recordsLinked to original sources

PubMed2003

Vitamin D regulated keratinocyte differentiation: role of coactivators.

1,25 Dihydroxyvitamin D (1,25(OH)(2)D) regulates the differentiation of keratinocytes. 1,25(OH)(2)D raises intracellular free calcium (Cai) as a necessary early step toward stimulating differentiation. 1,25(OH)(2)D induces the calcium sensing receptor (CaR) in keratinocytes and enhances the calcium response of these cells. Activation of the CaR by calcium increases intracellular free calcium by a mechanism involving phospholipase C (PLC) cleavage of phosphatidylinositolbisphosphate into inositoltrisphosphate (IP(3)) and diacylglycerol (DG). 1,25(OH)(2)D induces the family of PLCs. PLC-gamma1 has a DR6 VDRE in its promoter which binds and is activated by VDR/RAR rather than VDR/RXR. The involucrin gene, which encodes a critical component of the cornified envelope, contains a DR3 VDRE in its promoter that acts in conjunction with a nearby AP-1 site. The sequential regulation of these genes is critical for the differentiation process. In undifferentiated keratinocytes, the VDR binds preferentially to the DRIP complex of coactivators. However, with differentiation DRIP 205 is no longer produced, and the VDR switches partners to the SRC family (SRC2 and 3). These studies suggest that at least part of the sequential activation of genes required during keratinocyte differentiation is regulated by the change (availability) of these different coactivator complexes.

Animals↗

PubMed2003

Skeletal unloading induces resistance to insulin-like growth factor I on bone formation.

Skeletal unloading results in an inhibition of bone formation associated with a decrease in osteoblast number, impaired mineralization of bone, and altered proliferation and differentiation of osteoprogenitor cells. Although such changes are likely to be mediated by multiple factors, resistance to the growth-promoting action of insulin-like growth factor I (IGF-I) has been hypothesized to play an important role. To determine whether skeletal unloading induces resistance to IGF-I on bone formation, we examined the response of unloaded (hindlimb elevation) and normally loaded tibia and femur to IGF-I administration. To eliminate the variable of endogenous growth hormone production and secretion during exogenous IGF-I administration, we used growth hormone-deficient dwarf rats (dw-4). The rats were given IGF-I (2.5 mg/kg/day) or vehicle during 7 and 14 days of unloading or normal loading. This significantly increased the serum level of IGF-I in both the normally loaded and unloaded rats. Unloading did not affect the serum level of IGF-I in the vehicle-treated rats. IGF-I markedly increased periosteal bone formation at the tibiofibular junction of normally loaded rats. Unloading decreased bone formation in the vehicle-treated rats, and blocked the ability of IGF-I to increase bone formation. On the other hand, IGF-I increased periosteal bone formation at the midpoint of the humerus (normally loaded in this model) in both hindlimb-elevated and normally loaded rats. IGF-I significantly increased osteogenic colony number, total ALP activity, and total mineralization in bone marrow osteoprogenitor (BMOp) cells of normally loaded rats. Unloading reduced these parameters in the vehicle-treated rats, and blocked the stimulation by IGF-I. Furthermore, IGF-I administration (10 ng/ml) in vitro significantly increased cell proliferation of the BMOp cells isolated from normally loaded bone, but not that of cells from unloaded bone. These results indicate that skeletal unloading induces resistance to IGF-I on bone formation.

Animals↗

PubMed2002

Mechanical and chemical characteristics of mineral produced by basic fibroblast growth factor-treated bone marrow stromal cells in vitro.

It has been shown that various organ and cell cultures exhibit increased mineral formation with the addition of basic fibroblast growth factor (bFGF) and phosphate ions in the medium. However, to date there has been no attempt to relate the chemical composition of mineral formed in vitro to a measure of its mechanical properties. This information is important for understanding the in vivo mineralization process, the development of in vitro models, and the design of tissue-engineered bone substitutes. In this study we examined the reduced modulus; hardness; and mineral-to-matrix, crystallinity, carbonate-to-mineral, and calcium-to-phosphorus ratios of mineral formed by bFGF-treated rat-derived bone marrow stromal cells in vitro. The cells were treated with 1 or 3 mM beta-glycerophosphate for 3 and 4 weeks. Both mechanical parameters, reduced modulus and hardness, increased with increasing beta-glycerophosphate concentration. The only chemical measure of the mineral composition that exhibited the same dependency was the mineral-to-matrix ratio. The values of crystallinity and carbonate fraction were similar to those for intact cortical bone, but the calcium-to-phosphorus ratio was substantially lower than that of normal bone. These data indicate that the mineral formed by bFGF-treated bone cells is mechanically and chemically different from naturally formed lamellar bone tissue after 4 weeks in culture. These results can be used to improve in vitro models of mineral formation as well as enhance the design of tissue-engineered bone substitutes.

Animals↗

PubMed2001

The extracellular calcium-sensing receptor is required for calcium-induced differentiation in human keratinocytes.

In cultured keratinocytes, the acute increase of the extracellular calcium concentration above 0.03 mM leads to a rapid increase in intracellular calcium concentration ([Ca(2+)]i) and inositol trisphosphate production and, subsequently, to the expression of differentiation-related genes. Previous studies demonstrated that human keratinocytes express the full-length extracellular calcium-sensing receptor (CaR) and an alternatively spliced variant lacking exon 5 and suggested their involvement in calcium regulation of keratinocyte differentiation. To understand the role of the CaR, we transfected keratinocytes with an antisense human CaR cDNA construct and examined its impact on calcium signaling and calcium-induced differentiation. The antisense CaR cDNA significantly reduced the protein level of endogenous CaRs. These cells displayed a marked reduction in the rise in [Ca(2+)]i in response to extracellular calcium or to NPS R-467, a CaR activator, whereas the ATP-evoked rise in [Ca(2+)]i was not affected. Calcium-induced inhibition of cell proliferation and calcium-stimulated expression of the differentiation markers involucrin and transglutaminase were also blocked by the antisense CaR cDNA. When cotransfected with luciferase reporter vectors containing either the involucrin or transglutaminase promoter, the antisense CaR cDNA suppressed the calcium-stimulated promoter activities. These results indicate that CaR is required for mediating calcium signaling and calcium-induced differentiation in keratinocytes.

Amino Acid Sequence↗

PubMed2001

Calcium- and vitamin D-regulated keratinocyte differentiation.

Calcium and 1,25 dihydroxyvitamin D (1,25(OH)(2)D) regulate the differentiation of keratinocytes. We have examined the mechanisms by which such regulation takes place, focusing primarily on the events leading to cornified envelope (CE) formation, in particular the mechanisms by which calcium and 1,25(OH)(2)D regulate the induction of involucrin, a component of the CE, and transglutaminase, the enzyme cross-linking involucrin and other substrates to form the CE. Both extracellular calcium (Ca(o)) and 1,25(OH)(2)D raise intracellular free calcium (Ca(i)) as a necessary step toward stimulating differentiation. Cells lacking the calcium sensing receptor (CaR) or phospholipase C-gamma 1 (PLC-gamma 1) fail to respond to Ca(o) or 1,25(OH)(2)D with respect to differentiation. Residing in the promoter of involucrin is a region responsive to calcium and 1,25(OH)(2)D, the calcium response element (CaRE). The CaRE contains an AP-1 site, mutations of which result in loss of responsiveness to Ca(o) and 1,25(OH)(2)D, indicating a role for protein kinases C (PKC). PKC alpha is the major PKC isozyme involved at least for calcium-induced differentiation. Thus, the regulation of keratinocyte differentiation by calcium and 1,25(OH)(2)D involves a number of signaling pathways including PLC and PKC activation, leading to the induction of proteins required for the differentiation process.

Animals↗

PubMed2001

Inhibition of 1,25-dihydroxyvitamin-D-induced keratinocyte differentiation by blocking the expression of phospholipase C-gamma1.

Keratinocytes produce vitamin D3 and convert it to the most active form, 1,25-dihydroxyvitamin D3, which regulates keratinocyte proliferation and differentiation. Phospholipase C-gamma1 is the most abundant member of the phospholipase C family in keratinocytes and is induced by 1,25-dihydroxyvitamin D3. Therefore, phospholipase C-gamma1 might be important in the signaling pathway mediating 1,25-dihydroxyvitamin-D3-induced keratinocyte differentiation. To test this hypothesis, phospholipase C-gamma1 expression in human keratinocytes was reduced by transfecting the cells with an antisense phospholipase C-gamma1 construct and then evaluating the response of the keratinocyte differentiation markers involucrin and transglutaminase to 1,25-dihydroxyvitamin D3. The results showed that involucrin and transglutaminase protein and mRNA levels were markedly reduced in keratinocytes transfected by the antisense phospholipase C-gamma1 construct. Cotransfection of keratinocytes with the involucrin or transglutaminase promoter construct and the antisense phospholipase C-gamma1 construct showed decreased involucrin or transglutaminase promoter activity in response to 1,25-dihydroxyvitamin D3. To further investigate the mechanism by which phospholipase C-gamma1 regulates keratinocyte differentiation, the calcium and inositol triphosphate levels in keratinocytes transfected by the antisense phospholipase C-gamma1 construct were measured following 1,25-dihydroxyvitamin D3 administration. The increase in keratinocyte intracellular free calcium and inositol triphosphate levels following 1,25-dihydroxyvitamin D3 administration were markedly reduced by the transfection of the antisense phospholipase C-gamma1 construct. These studies indicate that phospholipase C-gamma1 plays a critical role in the signal transduction pathway mediating 1,25-dihydroxyvitamin-D3-induced keratinocyte differentiation at least in part by mediating the increase in inositol triphosphate production and intracellular calcium mobilization following 1,25-dihydroxyvitamin D3 administration.

Calcium↗

PubMed2001

Osteoblasts respond to pulsatile fluid flow with short-term increases in PGE(2) but no change in mineralization.

Although there is no consensus as to the precise nature of the mechanostimulatory signals imparted to the bone cells during remodeling, it has been postulated that deformation-induced fluid flow plays a role in the mechanotransduction pathway. In vitro, osteoblasts respond to fluid shear stress with an increase in PGE(2) production; however, the long-term effects of fluid shear stress on cell proliferation and differentiation have not been examined. The goal of this study was to apply continuous pulsatile fluid shear stresses to osteoblasts and determine whether the initial production of PGE(2) is associated with long-term biochemical changes. The acute response of bone cells to a pulsatile fluid shear stress (0.6 +/- 0.5 Pa, 3.0 Hz) was characterized by a transient fourfold increase in PGE(2) production. After 7 days of static culture (0 dyn/cm(2)) or low (0.06 +/- 0.05 Pa, 0.3 Hz) or high (0.6 +/- 0.5 Pa, 3.0 Hz) levels of pulsatile fluid shear stress, the bone cells responded with an 83% average increase in cell number, but no statistical difference (P > 0.53) between the groups was observed. Alkaline phosphatase activity per cell decreased in the static cultures but not in the low- or high-flow groups. Mineralization was also unaffected by the different levels of applied shear stress. Our results indicate that short-term changes in PGE(2) levels caused by pulsatile fluid flow are not associated with long-term changes in proliferation or mineralization of bone cells.

Animals↗

PubMed2001

Cholesterol sulfate stimulates involucrin transcription in keratinocytes by increasing Fra-1, Fra-2, and Jun D.

Lipids that are synthesized de novo in the epidermis, including fatty acids, oxysterols, 1,25-dihydroxyvitamin D(3), and farnesol, can regulate the differentiation of normal human keratinocytes (NHK). Cholesterol sulfate (CS), an epidermal lipid that is produced in the upper nucleated layers of the epidermis coincident with terminal differentiation, has been shown to play a role in the regulation of the late stages of keratinocyte differentiation, including formation of the cornified envelope. In the present study, we determined i) whether CS regulates involucrin (INV), an early keratinocyte differentiation marker, and ii) the mechanism by which CS regulates differentiation. mRNA and protein levels of INV, a precursor protein of the cornified envelope, increased 2- to 3-fold in NHK incubated in the presence of CS. In contrast, cholesterol had no effect on INV protein or mRNA levels. Transcriptional regulation was assessed in NHK transfected with INV promoter-luciferase constructs. CS increased luciferase reporter activity approximately 2- to 3-fold in NHK transfected with a 3.7-kb INV promoter construct. Deletional analysis revealed a CS-responsive region of the INV promoter located between bp --2452 and --1880. A 5-base pair (bp) mutation of the AP-1 site (bp --2117 to --2111) within this responsive region abolished CS responsiveness, suggesting a role for the AP-1 complex in the regulation of INV transcription by CS. Electrophoretic mobility shift analysis demonstrated increased binding of nuclear extracts isolated from CS-treated NHK to AP-1 DNA as compared with vehicle-treated controls. Incubation of the nuclear extract with the appropriate antibodies showed that the AP-1 DNA-binding complex contained Fra-1, Fra-2, and Jun D. Western blots demonstrated that CS treatment increased the levels of Fra-1, Fra-2, and Jun D, and Northern analyses revealed that CS increased mRNA levels for these same AP-1 factors. These data indicate that CS, an endogenous lipid synthesized by keratinocytes, regulates the early stages of keratinocyte differentiation, and may do so through its ability to modulate levels of AP-1 proteins. -- Hanley, K., L. Wood, D. C. Ng, S. S. He, P. Lau, A. Moser, P. M. Elias, D. D. Bikle, M. L. Williams, and K. R. Feingold. Cholesterol sulfate stimulates involucrin transcription in keratinocytes by increasing Fra-1, Fra-2, and Jun D. J. Lipid Res. 2001. 42: 390--398.

Binding Sites↗

PubMed2000

Requirement of an AP-1 site in the calcium response region of the involucrin promoter.

Involucrin is a major protein of the cornified envelope of keratinocytes that provides much of the structural integrity of the skin. The gene expression of this differentiation marker is induced by elevated extracellular calcium in cultured human keratinocytes. A 3.7-kilobase fragment of this gene contains the necessary elements to drive a luciferase reporter in a calcium-dependent manner. We have sequenced the upstream region of the involucrin promoter and localized a calcium response element that contains an activating protein-1 (AP-1) site (TGAGTCA). Mutation of this site abolished the promoter activation by calcium. Compared with cells grown in 0.03 mm calcium, the binding activity of factors within nuclear extracts from keratinocytes for this AP-1 site was enhanced 3-fold in cells grown in 1.2 mm calcium. Immunoelectrophoretic mobility shift (supershift) assays identified JunD, Fra1, and Fra2 as the major factors that bind to the AP-1 element. Western analysis of the proteins in the nuclear extracts showed that the levels of c-Jun, JunB, JunD, FosB, and Fra2 increased and the levels of c-Fos and Fra1 decreased slightly with calcium treatment. The effect of calcium on the involucrin promoter was enhanced synergistically by phorbol 12-myristate 13-acetate (PMA) in a protein kinase-dependent manner. In conclusion, calcium-regulated involucrin gene expression is mediated at least in part by AP-1 transcription factors.

Base Sequence↗

PubMed2000

Farnesol stimulates differentiation in epidermal keratinocytes via PPARalpha.

The isoprenoids farnesol and juvenile hormone III (JH), metabolites of the cholesterol biosynthetic pathway, have been shown to stimulate fetal epidermal development in rodents. In this study we determined whether this effect might be attributed to a direct induction of keratinocytes differentiation and examined the mechanisms responsible for these effects. Rates of cornified envelope formation, a marker of keratinocyte terminal differentiation, as well as protein and mRNA levels of two proteins required for cornified envelope formation, involucrin (INV) and transglutaminase, increased 2- to 3-fold in normal human keratinocytes (NHK) treated with either farnesol or JH, even at low calcium concentrations (0.03 mM), which otherwise inhibit differentiation. In contrast, neither cholesterol nor mevalonate affected INV or transglutaminase mRNA levels. Effects of farnesol and JH on INV and transglutaminase mRNA levels were additive with high calcium concentrations (1.2 mM) that independently stimulate keratinocyte differentiation. In contrast, keratinocyte DNA synthesis was inhibited by these compounds. Both farnesol and JH stimulated INV and transglutaminase promoter activity, suggesting regulation at the transcriptional level. A series of truncation and deletion experiments revealed a farnesol-responsive region (-2452 to -1880 base pairs (bp)) in the INV gene. This region contained an AP-1 site. A single base pair mutation of the AP-1 site at -2116 to -2110 bp abolished farnesol responsiveness, identical to effects by peroxisome proliferator-activated receptor (PPARalpha) activators. Farnesoid X-activated receptor mRNA was not detected in NHK, but farnesol treatment increased activities of both a PPAR response element and PPARalpha mRNA levels in NHK. Furthermore, the increase in PPRE activity by farnesol was dependent upon PPARalpha in CV-1 cells. Finally, topical applications of farnesol increased mRNA and protein levels of the differentiation-specific genes, profilaggrin and loricrin, determined by immunohistochemistry and in situ hybridization, in wild-type but not in PPARalpha-/- murine epidermis. These findings suggest a novel role for selected isoprenoid cholesterol intermediates in the regulation of differentiation-specific gene transcription and a convergence of PPARalpha with the cholesterol synthetic pathway.

Animals↗

PubMed2000

The calcium sensing receptor and its alternatively spliced form in murine epidermal differentiation.

We have recently reported that human keratinocytes express both the full-length calcium sensing receptor (CaR) and an alternatively spliced form lacking exon 5, which were suggested to be involved in calcium induced keratinocyte differentiation. To understand further the role of these CaRs, we analyzed the structure of mouse CaRs, and investigated their role using a mouse model in which only the full-length CaR was disrupted. Our results show that both the full-length and the alternatively spliced variant lacking exon 5 encoding 77 amino acids of the extracellular domain were expressed in mouse epidermis. The deletion of the full-length CaR increased the production of the alternatively spliced form of CaR in mutant mice. The keratinocytes derived from these mutant mice did not respond to extracellular calcium, suggesting that the full-length CaR is required to mediate calcium signaling in the keratinocytes. The loss of the full-length CaR altered the morphologic appearance of the epidermis and resulted in a reduction of the mRNA and protein levels of the keratinocyte differentiation marker, loricrin. These results indicate that CaR is important in epidermal differentiation, and that the alternatively spliced form does not fully compensate for loss of the full-length CaR.

Alternative Splicing↗

PubMed2000

Stimulation of PPARalpha promotes epidermal keratinocyte differentiation in vivo.

Our recent studies have demonstrated that PPARalpha activators stimulate differentiation and inhibit proliferation in cultured human keratinocytes and accelerate epidermal development and permeability barrier formation in fetal rat skin explants. As the role of PPARalpha activation in adult epidermis is not known, the aim of this study was to determine if topically applied PPARalpha ligands regulate keratinocyte differentiation in murine epidermis. Topical treatment with PPARalpha activators resulted in decreased epidermal thickness. Expression of structural proteins of the upper spinous/granular layers (involucrin, profilaggrin-filaggrin, loricrin) increased following topical treatment with PPARalpha activators. Furthermore, topically applied PPARalpha activators also increased apoptosis, decreased cell proliferation, and accelerated recovery of barrier function following acute barrier abrogation. Experiments with PPARalpha-/- knockout mice showed that these effects are specifically mediated via PPARalpha. Compared with the epidermis of PPARalpha+/+ mice, involucrin, profilaggrin-filaggrin, and loricrin expression were slightly decreased in PPARalpha-/- mice. Moreover, topical clofibrate treatment did not increase epidermal differentiation in PPARalpha-/- mice. Furthermore, in cultured human keratinocytes we have demonstrated that PPARalpha activators induce an increase in involucrin mRNA levels. We have also shown that this increase in gene expression requires an intact AP-1 response element at -2117 to -2111 bp. Thus, stimulation of PPARalpha stimulates keratinocyte/epidermal differentiation and inhibits proliferation.

Administration, Cutaneous↗

PubMed2000

Oxysterols induce differentiation in human keratinocytes and increase Ap-1-dependent involucrin transcription.

Ligands and activators of the nuclear hormone receptor superfamily are important in the regulation of epidermal development and differentiation. Previously, we showed that naturally occurring fatty acids, as well as synthetic ligands for the peroxisome proliferator-activated receptor, induce keratinocyte differentiation in vitro. Here we asked whether oxysterols, another class of lipids formed de novo in the epidermis and that activate liver X-activated receptor, regulate keratinocyte differentiation. mRNA and protein levels of involucrin and transglutaminase 1, markers of differentiation, increased 2- to 3-fold in normal human keratinocytes incubated in the presence of 25- or 22R-hydroxycholesterol in low calcium. In high calcium, which alone induces differentiation, mRNA levels were further increased by oxysterols. Rates of cornified envelope formation, an indicator of terminal differentiation, also increased 2-fold with oxysterol treatment. In contrast, the rate of DNA synthesis was inhibited approximately 50% by oxysterols. Transcriptional regulation was assessed in keratinocytes transfected with either transglutaminase 1 or involucrin promoter-luciferase constructs. 22R-hydroxycholesterol increased transglutaminase 1 and involucrin promoter activity 2- to 3-fold. Either deletion of the -2452 bp to -1880 bp region of the involucrin promoter, or mutation of the AP-1 site within this region, abolished oxysterol responsiveness. Moreover, increased AP-1 DNA binding was observed in oxysterol-treated keratinocytes by gel shift analyses. Finally, we demonstrated the presence of liver X-activated receptor alpha and beta mRNAs, and showed that oxysterols stimulate a liver X-activated receptor response element transfected into keratinocytes. These data suggest that oxysterols induce keratinocyte differentiation, in part through increased AP-1-dependent transcription of the involucrin gene, an effect that may be mediated by liver X-activated receptor.

Calcium↗

PubMed1999

Phospholipase C-gamma1 is required for calcium-induced keratinocyte differentiation.

Phospholipase C-gamma1 is the most abundant member of the phospholipase C family in keratinocytes and is induced by calcium. Phospholipase C-gamma1, therefore, may be involved in the signal transduction system leading to calcium regulation of keratinocyte differentiation. To test this hypothesis, expression of phospholipase C-gamma1 in human keratinocytes was blocked by transfecting cells with the antisense human phospholipase C-gamma1 cDNA construct. These cells demonstrated a dramatic reduction in phospholipase C-gamma1 protein level compared with the empty vector-transfected cells and a marked reduction in the mRNA and protein levels of the differentiation markers involucrin and transglutaminase following administration of calcium. Similarly, cotransfection of antisense phospholipase C-gamma1 constructs with a luciferase reporter vector containing involucrin or transglutaminase promoters led to a substantial reduction in calcium-stimulated involucrin and transglutaminase promoter activities. Similar results were seen following treatment with a specific phospholipase C inhibitor U73122. To determine whether phospholipase C-gamma1 regulated differentiation by controlling intracellular calcium, we examined the ability of antisense phospholipase C-gamma1 to block the calcium-induced rise in intracellular calcium and found that it could. These findings indicate that phospholipase C-gamma1 is a critical component of the signaling pathway mediating calcium regulation of keratinocyte differentiation via its mobilization of intracellular calcium.

Blotting, Western↗

PubMed1999

Six putative IQ motifs of the recombinant chicken intestinal brush border myosin I are involved in calmodulin binding.

Chicken brush border myosin I has up to six IQ sequence motifs at which it may bind calmodulin. To determine the relative contributions of these motifs to calmodulin binding, fusion deletion fragments were expressed in Escherichia coli and their ability to bind calmodulin was assessed by the gel overlay technique. The first three N-terminal IQ sites showed strong binding with calmodulin. Surprisingly, the last three incomplete IQ motifs also contributed substantial calmodulin binding. The first and fourth IQ sites bound calmodulin but tended to reduce binding in combination with other sites. The data indicate that interactions among all six IQ motifs contribute to the ability of myosin I to bind calmodulin.

Animals↗

PubMed1999

1,25 dihydroxyvitamin D3 enhances the calcium response of keratinocytes.

The steroid hormone 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) regulates cell proliferation and differentiation. Intracellular calcium (Cai) concentrations play a crucial role in these events. From our previous studies, we have demonstrated a calcium receptor (CaR) in keratinocytes which appears to regulate the initial release of Cai from intracellular stores in response to extracellular calcium (Cao) and so is likely to participate in the differentiation process. In this study, we determined whether the ability of 1,25(OH)2D3 to enhance Ca++ -induced differentiation was mediated at least in part through changes in the CaR. Keratinocytes were grown in keratinocyte growth medium (KGM) with 0.03 mM, 0.1 mM, or 1.2 mM Ca and treated with 10(-8) M 1,25(OH)2D3 till harvest after 5, 7, 14, and 21 days. CaR mRNA levels were quantitated by polymerase chain reaction. The results were compared to the ability of 1,25(OH)2D3 to enhance calcium-stimulated increases in Cai. In cells grown in 0.03 mM Ca, the CaR mRNA levels decreased with time. 1,25(OH)2D3 stimulated the levels at 5 days and prevented the falloff over the subsequent 16 days. On the other hand, in cells grown in 0.1 or 1.2 mM Ca, the message levels remained high, and 1,25(OH)2D3 had no further effect. To study the functional relationship, we harvested cells after 5 and 7 days in culture following a 24 h treatment with 1,25(OH)2D3 or vehicle to measure the Cai response to 2 mM Cao. The preconfluent cells grown in 0.03 mM Ca showed a nearly twofold increase in the Cai response to Cao when pretreated with 1,25(OH)2D3, whereas the confluent cells and those grown in 1.2 mM Ca showed no enhancement by 1,25(OH)2D3. Studies with 45Ca influx into keratinocytes revealed that 1,25(OH)2D3 enhanced the influx in preconfluent and confluent cells when grown in KGM containing 0.03 mM Ca but not in cells grown in 1.2 mM calcium. We conclude that 1,25(OH)2D3 maintains the CaR mRNA levels in cells grown in 0.03 mM Ca, thus maintaining their responsiveness to Cao and so ensuring their ability to differentiate in response to the calcium signal.

Base Sequence↗

PubMed1999

The response of bone to unloading.

Skeletal unloading leads to decreased bone formation and decreased bone mass. Bone resorption is uncoupled from bone formation, contributing to the bone loss. During spaceflight bone is lost principally from the bones most loaded in the 1-g environment, and some redistribution of bone from the lower extremities to the head appears to take place. Although changes in calcitropic hormones have been demonstrated during skeletal unloading (PTH and 1,25(OH)2D decrease), it remains unclear whether such changes account for or are in response to the changes in bone formation and resorption. Bed rest studies with human volunteers and hindlimb elevation studies with rats have provided useful data to help explain the changes in bone formation during spaceflight. These models of skeletal unloading reproduce a number of the conditions associated with microgravity, and the findings from such studies confirm many of the observations made during spaceflight. Determining the mechanism(s) by which loading of bone is sensed and translated into a signal(s) controlling bone formation remains the holy grail in this field. Such investigations couple biophysics to biochemistry to cell and molecular biology. Although studies with cell cultures have revealed biochemical responses to mechanical loads comparable to that seen in intact bone, it seems likely that matrix-cell interactions underlie much of the mechanocoupling. The role for systemic hormones such as PTH, GH, and 1,25(OH)2D compared to locally produced factors such as IGF-I, PTHrP, BMPs, and TGF-beta in modulating the cellular response to load remains unclear. As the mechanism(s) by which bone responds to mechanical load with increased bone formation are further elucidated, applications of this knowledge to other etiologies of osteoporosis are likely to develop. Skeletal unloading provides a perturbation in bone mineral homeostasis that can be used to understand the mechanisms by which bone mineral homeostasis is maintained, with the expectation that such understanding will lead to effective treatment for disuse osteoporosis.

Animals↗

PubMed1999

Effects of a calcium receptor activator on the cellular response to calcium in human keratinocytes.

Changes in the concentration of extracellular calcium affect the balance between proliferation and differentiation in epidermal keratinocytes. Undifferentiated keratinocytes respond to the acute increase in the concentration of extracellular calcium with an increase of intracellular calcium concentration and inositol trisphosphate production, and, subsequently, the expression of differentiation related genes. Our previous studies demonstrated the presence of a calcium-sensing receptor in human keratinocytes, which is identical to the parathyroid calcium-sensing receptor. In this study we showed that the calcimimetic compound NPS R-467, a selective calcium-sensing receptor activator, augmented the calcium-elicited inositol trisphosphate response of cloned human keratinocyte calcium-sensing receptor expressed in human embryonic kidney cells 293. In order to define the role of the calcium-sensing receptor in calcium induced epidermal differentiation, we investigated the ability of NPS R-467 to raise intracellular Ca2+ and stimulate differentiation in normal human foreskin keratinocytes. In the presence of 0.03 mM Ca2+, NPS R-467 increased the intracellular calcium concentration response in a concentration-dependent fashion. Undifferentiated normal human foreskin keratinocyte cells responded to increased extracellular calcium concentration with increased intracellular calcium concentration. NPS R-467 potentiated this response by increasing the maximal response. Its stereoisomer, NPS S-467, was not active in raising intracellular calcium concentration. Increasing extracellular calcium concentration from 0.03 to 1.2 mM stimulated the promoter activity of the differentiation marker gene, involucrin. NPS R-467 potentiated the calcium-stimulated increase in involucrin promoter activity unlike NPS S-467 or vehicle. Northern analysis of the normal human foreskin keratinocyte cells treated with NPS R-467 demonstrated potentiation of the calcium-stimulated increases in involucrin and transglutaminase mRNA levels. These results support the hypothesis that the calcium-sensing receptor expressed in keratinocytes mediates at least part of the intracellular calcium response to extracellular calcium and calcium-induced differentiation.

Aniline Compounds↗