The role of protein kinase C-delta in PTH stimulation of IGF-binding protein-5 mRNA in UMR-106-01 cells

Mary S. Erclik and Jane Mitchell

Department of Pharmacology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have investigated the role of protein kinase C (PKC) signal transduction pathways in parathyroid hormone (PTH) regulation of insulin-like growth factor-binding protein-5 (IGFBP-5) gene expression in the rat osteoblast-like cell line UMR-106-01. Involvement of the PKC pathway was determined by the findings that bisindolylmaleimide I inhibited 40% of the PTH effect, and 1 µM bovine PTH-(3-34) stimulated a 10-fold induction of IGFBP-5 mRNA. PTH-(1-34) and PTH-(3-34) (100 nM) both stimulated PKC-delta translocation from the membrane to the nuclear fraction. Rottlerin, a PKC-delta -specific inhibitor, and a dominant negative mutant of PKC-delta were both able to significantly inhibit PTH-(1-34) and PTH-(3-34) induction of IGFBP-5 mRNA, suggesting a stimulatory role for PKC-delta in the effects of PTH. Phorbol 12-myristate 13-acetate (PMA) stimulated PKC-alpha translocation from the cytosol to the membrane and inhibited ~50% of the PTH-(1-34), forskolin, and 8-bromoadenosine 3',5'-cyclic monophosphate-stimulated IGFBP-5 mRNA levels, suggesting that PKC-alpha negatively regulates protein kinase A (PKA)-mediated induction of IGFBP-5 mRNA. These results suggest that the induction of IGFBP-5 by PTH is both PKA and PKC dependent and PKC-delta is the primary mediator of the effects of PTH via the PKC pathway.

insulin-like growth factor-binding protein-5; osteoblast; protein kinase C; messenger RNA; protein kinase A


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PARATHYROID HORMONE (PTH) is an essential mediator of extracellular calcium homeostasis. Its effects in vivo influence bone turnover to favor bone resorption (11). However, PTH can also have anabolic effects on bone if administered intermittently at low doses and is presently in clinical testing for the treatment of osteoporosis (24). The effects of PTH in vivo are also observed in vitro with osteoblasts in culture and are thought to result from the PTH/PTH-related peptide receptor's ability both to couple to Gsalpha and stimulate protein kinase A (PKA) and to couple to Gq/11alpha and stimulate protein kinase C (PKC) (12, 18, 27). The concomitant stimulation of the PKA and PKC pathway upon PTH-receptor activation by intact PTH mediates its cellular effects.

PTH has been shown to stimulate the production of insulin-like growth factors (IGFs) and insulin-like growth factor-binding proteins (IGFBPs) (4, 20). IGFs are important regulators of osteoblast metabolism, and their effects include stimulation of collagen production and mitogenesis (9, 16, 21). IGFBPs are thought to regulate the effects of IGFs, and IGFBP-5 specifically has been shown to potentiate the anabolic effects of IGFs, possibly by sequestering IGFs in proximity to their membrane receptors (16). IGFBP-5 has also been shown to act as an osteoblast mitogen independently of IGF, albeit with less potency (1). The effects of IGFs and IGFBP-5 may therefore contribute to the anabolic actions of PTH on bone.

Previous studies have demonstrated that pharmacological agents that stimulate the PKA pathway, such as forskolin and (Bu)2AMP, are able to induce IGFBP-5 transcript levels in both skin fibroblasts and osteoblastic cells (2, 4). Further studies in fibroblasts have indicated a role for activating protein (AP)-2 transactivation in cAMP responsiveness of the IGFBP-5 gene (5), although AP-2 does not appear to mediate PGE2 stimulation of IGFBP-5 transcription in primary osteoblasts (14). The PKC pathway has also been implicated in the stimulation of IGFBP-5 in human fibroblasts (6), and one study utilizing PTH analogs suggested that a pathway other than adenylyl cyclase mediated part of the PTH effect on IGFBP-5 mRNA in osteoblasts (25). It is not clear which PKC isozyme(s) is responsible for the regulation of IGFBP-5 in any of these cell types.

The PKC gene family consists of three subgroups based on sequence similarity and cofactor dependence: classical PKCs (alpha , beta I, beta II, and gamma ), novel PKCs (delta , epsilon , eta , and theta ) and atypical PKCs (zeta  and iota ). Once activated, PKC isozymes translocate to specific cellular compartments, a process that is both isozyme and cell-type specific. Each isozyme is then capable of interacting with its own specific substrates and subsequently mediate distinct cellular events (23, 35). Thus it is important to identify the specific PKC isozymes that mediate signal transduction events, because their effects in many instances are quite distinct.

To further characterize a role for PKC isozymes in the regulation of IGFBP-5, in the present study, we have used the well characterized osteoblastic UMR-106-01 cell line that we and others have used extensively to study osteoblast regulation (22, 33, 34). IGFBP-5 mRNA levels are extremely low in this cell line in the basal state and are increased by stimulation with PTH. We report that PTH selectively regulates the cellular distribution of PKC-delta and that its activation, along with PKA, mediates PTH induction of IGFBP-5 mRNA.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. UMR-106-01 cells (a generous gift from Dr. N. Partridge, St. Louis University, St. Louis, MO) were grown in 50% Dulbecco's modified Eagle's medium and 50% F-12 medium containing 1 U/ml penicillin, 1 µg/ml streptomycin, and 0.25 µg/ml amphotericin B and supplemented with 5% fetal calf serum (Life Technologies, Burlington, ON, Canada).

Materials. Rat PTH-(1-34), bovine PTH-(1-84), human PTH-(1-31), and bovine PTH-(3-34) were purchased from Bachem Bioscience (King of Prussia, PA). Bisindolylmaleimide I, rottlerin, phorbol 12-myristate 13-acetate (PMA), H-89, forskolin, and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) were purchased from Biomol (Plymouth Meeting, PA). Isozyme-specific PKC antibodies were purchased from BD Transduction Laboratories (Mississauga, ON, Canada).

Cell treatment and cellular fractionation. After 20-24 h of serum starvation, cells were treated with PTH analogs or PMA (in DMSO) in serum-free media containing 0.1% BSA for 30 min. Cytosol, membrane, and nuclear fractions of cells were prepared by previously described procedures with minor modifications (10). Briefly, cells were washed once with cold PBS and scraped from the plate on ice in 750 µl of buffer A [50 mM Tris · HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor, 500 µM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 1 mM NaF, and 1 mM sodium orthovanadate]. Cells were allowed to swell on ice for 1 h and were homogenized with 20-60 strokes of a Dounce homogenizer (pestle A) until >80% of the cells were lysed. Homogenates were centrifuged at 600 g for 20 min at 4°C to pellet a crude nuclear fraction. The supernatant was recentrifuged at 100,000 g for 30 min at 4°C to pellet membranes and the supernatant containing the cytosol. The membrane pellet was solubilized in buffer A containing 1% Triton X-100. The crude nuclear pellet was resuspended in buffer A containing 0.1% Triton X-100, layered over 30% sucrose (wt/vol in buffer A), and centrifuged at 5,000 g for 30 min at 4°C. The pellet was resuspended in buffer A containing 1% Triton X-100 and sonicated on ice for 15 s. In one experiment, total cell homogenate was prepared in the following way. Cells were transfected with respective constructs and, 24 h later, were washed once with cold PBS and scraped from the plate on ice with a rubber scraper in 750 µl of buffer B [50 mM Tris · HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 150 mM NaCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor, 500 µM AEBSF, 1 mM NaF, 1 mM sodium orthovanadate, 1% (vol/vol) 2-mercaptoethanol, 1% (vol/vol) NP-40, and 0.3% sodium deoxycholate]. Lysates were then sonicated on ice for 15 s. Protein concentrations were determined by the Amido Black method (30).

Western blotting of subcellular fractions. Equal amounts of protein (25-50 µg) of each fraction were separated on 11% SDS-polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes overnight at 4°C. Membranes were blocked for 1 h in PBS-0.2% Triton X-100 (PBST) containing 3% BSA. Membranes were then immunoblotted with monoclonal anti-isozyme-specific PKC antibodies in PBST and then probed with horseradish peroxidase-conjugated rabbit anti-mouse IgG antiserum, washed, and detected using an enhanced chemiluminescence detection reagent (Amersham Pharmacia, Baie d'Urfe, QC, Canada) on Kodak X-100 ARS film.

Northern and slot blot analysis. After 20-24 h of serum starvation in media containing 0.1% BSA, UMR-106-01 cells were treated for 6 h with PTH or other agents, and total cellular RNA was isolated with TRIzol reagent (Life Technologies, Burlington, ON, Canada). Having verified by Northern blot that both the IGFBP-5 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes recognized single transcripts, size 6.0 and 1.3 kb, respectively, we subsequently employed slot blots to assess transcript levels. Samples (3 µg) of total RNA were directly blotted onto a nylon membrane by means of a slot blot apparatus and then ultraviolet cross-linked. Blots were prehybridized for >= 1 h in 0.1 M sodium phosphate buffer containing 0.1% BSA, 9 mg/ml salmon sperm DNA, and 7% SDS at 65°C. The 300-bp IGFBP-5 cDNA probe was obtained by digesting IGFBP-5 cDNA (kindly provided by Dr. S. Shimasaki, The Whittier Institute for Diabetes and Endocrinology, La Jolla, CA) with HindIII and SacII (31). The probe was labeled with [alpha -32P]dCTP by use of the random hexanucleotide-primed method (7). Hybridizations were carried out in the prehybridization solution overnight at 65°C, and washes were performed at 65°C in 30 mM sodium phosphate buffer containing 0.1% SDS. As an internal control, parallel blots were prepared and hybridized under the same conditions with a 950-bp GAPDH cDNA probe, obtained by digesting GAPDH cDNA with SflI and BstEII, as previously described (8). Bound RNA was visualized by autoradiography on Kodak X-AR5 film. Signals were quantitated from a phosphorimager using ImageQuant.

Transient transfections. The mouse dominant negative PKC-delta K376R (DNPKC-delta ), and PKC-epsilon K376R (DNPKC-epsilon ) constructs were kindly provided by Dr. I. B. Weinstein (Columbia University, New York, NY) (32). Cells were grown to 60-70% confluence in 6-well plates over 48 h and transfected with DNPKC-delta , DNPKC-epsilon , or vector alone (pcDNA 3.1+) with Lipofectamine reagent (Life Technologies, Burlington, ON, Canada). Two micrograms of DNA and 8 µl of transfection reagent were used for each well in serum-free media. After a 24-h incubation, cells were treated with PTH or other agents for an additional 6 h, and then RNA was isolated and assessed as outlined. Transfection rates of ~30% were found using this protocol and were assessed as follows. Cells were transfected with the plasmid pSV-beta -gal (Promega, Madison, WI), which encodes the reporter gene beta -galactosidase. Twenty-four hours after transfection, cells were fixed in 3% formaldehyde (in PBS) for 15 min at room temperature, washed three times with PBS, and then stained with X-gal-containing solution (in PBS: 4 mM K+ ferrocyanate, 4 mM K+ ferricyanate, 4 mM MgCl2, and 0.4 mg/ml X-gal in dimethyl fluoride) for 2 h at 37°C. Transfection rates were determined as the percentage of blue-stained cells compared with the total number of cells.

Presentation of data and statistical analysis. The slot blots presented are representative of at least three experiments. The quantitated values of IGFBP-5 mRNA obtained from individual samples were corrected for GAPDH. Statistical analysis was performed by Student's t-test. The results are expressed as means ± SD.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanisms of PTH-mediated induction of IGFBP-5 mRNA. Previous studies suggested that part of the induction by PTH of IGFBP-5 mRNA in osteoblasts occurs through pathways other than PKA; however, the involvement of the PKC pathway has not been investigated. We utilized PTH fragments that selectively stimulate the PKC or PKA-plus-PKC pathways to determine their relative contribution to PTH effects on IGFBP-5 transcript. As demonstrated in Fig. 1A, under basal conditions, there were very low levels of IGFBP-5 transcript as assessed by phosphorimaging. PTH-(1-34), PTH-(1-84), and PTH-(1-31), each of which can stimulate PKA and PKC, all stimulated a 30- to 45-fold induction of IGFBP-5 mRNA levels. PTH-(3-34), an analog that has been shown to stimulate PKC but not PKA, stimulated a 5- to 20-fold induction of IGFBP-5, suggesting that PKC activation mediates part of PTH stimulation of IGFBP-5 transcript levels.


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Fig. 1.   Regulation of insulin-like growth factor-binding protein-5 (IGFBP-5) mRNA levels by regulators of the protein kinase A (PKA) and protein kinase C (PKC) pathways. A: UMR-106-01 cells were treated for 6 h with 10 nM parathyroid hormone rat (r): PTH-(1-34), bovine (b)PTH-(1-84), human (h)PTH-(1-31) or with 1 µM bPTH- (3-34). Equal amounts of total RNA were then subjected to slot blot analysis and hybridized with either an IGFBP-5 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. B: UMR-106-01 cells were treated with 10 nM PTH-(1-34), 5 µM forskolin, 1 µM phorbol 12-myristate 13-acetate (PMA) alone or cotreated with PTH and 20 µM H-89 or 5.0 µM bisindolylmaleimide I. Values obtained from blots hybridized with IGFBP-5 probe were corrected for GAPDH signals. Values are expressed as degree of stimulation of nontreated (control) cells. Bars represent means ± SD from >= 3 experiments. *Values significantly different (P < 0.01) from unstimulated (control) cells. #Values significantly different (P < 0.01) from PTH-(1-34) alone.

To further characterize the role of the PKC pathway in PTH induction of IGFBP-5 transcript, cells were treated with either PTH-(1-34) alone or the selective PKA and PKC inhibitors H-89 and bisindolylmaleimide I, respectively. Both inhibitors on their own had no effect on IGFBP-5 transcript levels (data not shown); however, 20 µM H-89 reduced PTH stimulation of IGFBP-5 mRNA from 45-fold to just 17-fold. When cells were cotreated with PTH-(1-34) and 5 µM bisindolylmaleimide I, PTH induction of IGFBP-5 was significantly reduced to just under 30-fold. Figure 1B shows that selective activation of adenylyl cyclase with forskolin resulted in a 25-fold induction of IGFBP-5 transcript levels, and this was inhibited in the presence of 20 µM H-89 to only fourfold (data not shown). Activation of PKC with 1 µM PMA, however, had no effect (Fig. 1B).

Effects of PTH-(1-34), PTH-(3-34), and PMA on PKC isozyme cellular distribution. Our initial findings suggested that IGFBP-5 mRNA is stimulated by PTH partially via the PKC pathway. However, direct activation of PKC by PMA had no effect. Because there are 11 members of the PKC family and only a subset of these is stimulated by PMA, we wished to determine which isozymes were activated by PTH or PMA.

Activated PKC isozymes have been shown to translocate from one intracellular location to another (26). Therefore, translocation can be used to identify which isozyme(s) could mediate the PTH downstream effects. Cytosol, membrane, and nuclear fractions of UMR-106-01 cells that were untreated (control) or incubated with PTH-(1-34), PTH-(3-34), or PMA were prepared, and the quantity of PKC isozymes present in each fraction was assessed by immunoblotting with isozyme-specific antibodies. Figure 2 demonstrates the distribution of representatives of each of the PKC subfamilies (PKC-alpha , classical, PKC-epsilon and PKC-delta , novel, and PKC-iota , atypical). Quantitative analysis of the translocation of each PKC isozyme is shown in Table 1.


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Fig. 2.   Regulation of the cellular distribution of PKC isozymes by PTH and PMA in UMR-106-01 cells. Cells were treated with either 100 nM rPTH-(1-34) or bPTH-(3-34) or 1 µM PMA for 30 min, and then cytosolic (C), membrane (M), and nuclear (N) fractions were prepared and immunoblotted with antibodies specific for PKC-alpha (A), PKC-delta and PKC-epsilon (B), and PKC-iota (C). Blots are representative of 3 independent experiments.


                              
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Table 1.   Quantitative analysis of PKC isozyme translocation in response to PMA, PTH-(1-34), and PTH-(3-34)

PKC-alpha migrates at an approximate molecular mass of 80 kDa and is predominantly found in the cytosol in resting cells. PTH-(1-34) or PTH-(3-34) failed to stimulate any change in the quantity or distribution of PKC-alpha . However, incubation with 1 µM PMA for 30 min stimulated the translocation of 54% of PKC-alpha from the cytosol to the membrane and 17% to the nuclear fractions. This effect was seen as early as 10 min after stimulation and was sustained for almost 1 h (data not shown).

PKC-delta was predominantly present in the membrane fraction and was identified as a doublet migrating at 75 kDa, whereas PKC-epsilon was localized in the cytosol and membrane fractions in resting cells and migrated also as a doublet at an approximate molecular mass of 90 kDa (Fig. 2B). The appearance of these two PKC isozymes as doublets has been noted previously and may result from the presence of both phosphorylated and unphosphorylated forms of the proteins (17). PTH-(1-34) stimulated the translocation of 45% of PKC-delta from the membrane to the nuclear fraction, leaving only 13% of the isozyme in the membrane. Likewise, PTH-(3-34) was able to stimulate PKC-delta translocation to the nucleus. PMA did not affect PKC-delta translocation; however, it did reduce the abundance of this isozyme by ~60% after a 30-min incubation. Downregulation of PKC-delta was seen as early as 20 min after addition of PMA and was sustained for at least 1 h (data not shown). PTH-(1-34), PTH-(3-34), and PMA all had no effect on the distribution of the second of the two novel PKC isozymes, PKC-epsilon ; however, PTH-(1-34) did decrease the total amount of PKC-epsilon by ~40%.

The atypical PKC isozyme PKC-iota was predominantly localized to the cytosol and membrane fractions in resting cells and migrated at an approximate molecular mass of 65 kDa. Neither PTH-(1-34) nor PMA had any effect on the subcellular distribution or amount of this isozyme (Fig. 2C).

Further examination of the time course of PTH-mediated translocation of PKC-delta demonstrated that, within 10 min of incubation with PTH-(1-34), PKC-delta began to translocate to the nucleus (Fig. 3). PKC-delta levels in the nucleus were maximal after 20 min, when 53% of this isozyme was associated with the nuclear fraction, and this was sustained for the next 10 min. After 40-50 min of incubation with PTH, PKC-delta began to move back to the membrane, returning to the basal distribution after 50 min (Fig. 3). Over this 50-min time course of PTH stimulation, the distribution of PKC-alpha and PKC-epsilon was unchanged (Fig. 3).


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Fig. 3.   Time course for PTH-(1-34) regulation of PKC isozyme cellular distribution. Cells were treated with either vehicle (0') or 100 nM rPTH- (1-34) for 10-50 min. Cytosolic (C), membrane (M), and nuclear (N) fractions were prepared and immunoblotted with antibodies specific for PKC-delta , PKC-alpha , or PKC-epsilon .

Involvement of PKC-delta in PTH-mediated stimulation of IGFBP-5 mRNA. Having established the ability of PTH to regulate the cellular distribution of PKC-delta , we next investigated whether this isozyme mediated part of the PTH induction of IGFBP-5 transcript. Cells were treated with PTH-(1-34) or PTH-(3-34) alone or in the presence of the PKC-delta -specific inhibitor rottlerin. When administered alone, rottlerin had no effect on IGFBP-5 mRNA levels (data not shown). Increasing concentrations of rottlerin progressively suppressed PTH-(1-34) induction of IGFBP-5, such that 5 µM rottlerin inhibited ~45% of the PTH signal (Fig. 4A). Rottlerin was similarly able to inhibit PTH-(3-34) induction of IGFBP-5 mRNA (Fig. 4B).


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Fig. 4.   Effect of the PKC-delta specific inhibitor rottlerin on rPTH-(1-34) and bPTH-(3-34)-stimulated induction of IGFBP-5 mRNA levels. A: UMR-106-01 cells were treated for 6 h with 10 nM PTH alone or in the presence of the indicated concentrations of rottlerin. Equal amounts of total RNA were then subjected to slot blot analysis and hybridized with either an IGFBP-5 or a GAPDH cDNA probe. B: cells were treated with 1 µM PTH-(3-34) alone or in the presence of indicated concentrations of rottlerin. Values obtained from blots hybridized with the IGFBP-5 probe were corrected for GAPDH. Values are expressed as degree of stimulation of nontreated (control) cells. Bars represent means ± SD from >= 3 experiments. *Values significantly different (P < 0.05) from PTH-(1-34) alone. #Values significantly different (P < 0.05) from PTH-(3-34) alone.

To further substantiate the involvement of PKC-delta in mediating PTH effects on IGFBP-5 transcript levels, PKC-delta activity was inhibited by a dominant negative mutant. The K376R mutation has been shown to eliminate the function of PKC-delta and is able to function as a dominant negative when transfected into cultured cells (19). DNPKC-delta or vector alone (pcDNA3.1) was transfected into UMR-106-01 cells, and 24 h later, cells were treated with PTH-(1-34) or PTH-(3-34). As shown in Fig. 5A, DNPKC-delta was able to inhibit the induction of IGFBP-5 stimulated by 10 nM PTH-(1-34) by ~30% compared with cells transfected with vector alone. When cells were transfected with DNPKC-epsilon , which also contained the K376R mutation and has previously been shown to act as a potent dominant negative mutant (32), the PTH induction of IGFBP-5 transcript was unaffected (Fig. 5A). DNPKC-delta was also able to partially block the PTH-(3-34) induction of IGFBP-5 mRNA by 25% compared with cells transfected with vector alone (Fig. 5B). Western blots of extracts from control cells or those transfected with DNPKC-delta or DNPKC-epsilon are shown in Fig. 5C and demonstrate the increased expression of these PKC isozymes in the transfected cells.


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Fig. 5.   Effect of dominant negative PKC-delta on rPTH-(1-34) and bPTH-(3-34) stimulation of IGFBP-5 mRNA. A: UMR-106-01 cells were transfected with either dominant negative PKC-delta (DNPKC-delta ), vector alone (pcDNA), or dominant negative PKC-epsilon (DNPKC-epsilon ) for 24 h. After 6-h treatment with 10 nM PTH-(1-34), total RNA was extracted, subjected to slot blot analysis, and hybridized with either an IGFBP-5 or a GAPDH cDNA probe. B: cells were transfected with either DNPKC-delta or vector alone and treated with 1 µM PTH-(3-34), and mRNA levels were examined. Bars represent means ± SD from >= 3 experiments. Values obtained from blots hybridized with IGFBP-5 probe were corrected for GAPDH. Values were expressed as degree of stimulation of nontreated (control) cells. *Values significantly different (P < 0.05) from PTH-(1-34) + vector alone. #Values significantly different (P < 0.01) from PTH-(3-34) + vector alone. C: expression levels of DNPKC-delta and DNPKC-epsilon in UMR-106-01 cells. Cells were either not transfected (control) or transfected with either DNPKC-delta , DNPKC-epsilon , or pcDNA3.1 for 24 h, and then 18 µg of total cell lysate were separated by PAGE. Figures on the left and right represent blots probed with PKC-delta - and PKC-epsilon -specific antibodies, respectively.

Effect of PMA on PKA-induced stimulation of IGFBP-5 mRNA. Our PKC isozyme translocation studies indicated that PMA stimulated the translocation of PKC-alpha and downregulated PKC-delta . The lack of effect of PMA alone on IGFBP-5 mRNA levels suggested that PKC-alpha does not stimulate IGFBP-5 transcription (Fig. 1). We also investigated whether PMA could affect IGFBP-5 gene expression stimulated by PKA. UMR-106-01 cells were treated with 5 µM forskolin, 1 mM 8-BrcAMP, or 10 nM PTH-(1-34) for 6 h in the presence or absence of 1 µM PMA. PMA blocked 50% of PTH-, forskolin-, and 8-BrcAMP-induced stimulation of IGFBP-5 transcript (Fig. 6).


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Fig. 6.   Effect of PMA on PTH-(1-34) and PKA-stimulated induction of IGFBP-5 mRNA. UMR-106-01 cells were treated with 10 nM rPTH-(1-34), 5 µM forskolin, or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) either alone or in the presence of 1 µM PMA. After 6 h of treatment, total RNA was extracted, subjected to slot blot analysis, and hybridized with an IGFBP-5 or a GAPDH cDNA probe. Bars represent means ± SD from >= 3 experiments. Values obtained from blots hybridized with the IGFBP-5 probe were corrected for GAPDH. Values were expressed as degree of stimulation of nontreated (control) cells. *Values significantly different (P < 0.01) from PTH-(1-34) alone. #Values significantly different (P < 0.01) from forskolin alone. **Values significantly different (P < 0.05) from 8-BrcAMP alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present report, we have demonstrated that both the PKA and PKC pathways mediate PTH stimulation of IGFBP-5 mRNA levels in UMR-106-01 osteoblastic cells. The functional involvement of PKA in PTH induction of IGFBP-5 is demonstrated in the present study by partial inhibition in the presence of H-89. However, this PKA inhibitor could not suppress 40% of PTH activation of IGFBP-5. Evidence that the PKC pathway mediated this additional PTH-stimulated IGFBP-5 came from several observations. First, the selective PKC inhibitor bisindolylmaleimide I partially blocked PTH stimulation of IGFBP-5. Second, PTH-(3-34), which is not able to stimulate adenylyl cyclase in UMR cells, was able to significantly stimulate IGFBP-5.

Because there was very little evidence for PKC regulation of IGFBP-5 transcription in the literature, we proceeded to investigate the regulation of the activity of PKC isozymes in UMR-106-01 cells. It has been reported by Sanders and Stern (29) that the related UMR-106 cell line expresses eight PKC isoforms, including PKC-alpha , -beta I, -beta II, -delta , -epsilon , -eta , -iota , and-zeta . We were unable to detect expression of either the beta - or eta -isoforms in the 106-01 cells, likely due to differences in the cell lines or the antibodies used in the two studies. We found that PTH-(1-34) and PTH-(3-34) had no effect on the distribution of PKC-alpha , -epsilon , and -iota but were able to stimulate translocation of PKC-delta to the nucleus, with a corresponding decrease in the membrane fraction over a 10- to 40-min time course. Others have previously reported that PKC-delta is translocated to the nucleus, in a number of different cell lines. For example, Wang et al. (35) demonstrated by means of fluorescence microscopy that bryostatin stimulated the rapid translocation of PKC-delta to the nucleus in Chinese hamster ovary cells. Translocation occurs after PKC activation, and peptide inhibitors of PKC isozyme translocation act as antagonists of PKC activity (15). To directly demonstrate the involvement of PKC-delta in PTH-(1-34) stimulation of IGFBP-5, we blocked the kinase activity of PKC-delta with the selective inhibitor rottlerin at concentrations between 0.5 and 5 µM. Rottlerin is a natural toxin that has been shown to inhibit PKC-delta with an IC50 of 3-6 µM by interaction with the ATP-binding domain (36). Other PKC isozymes and PKA are inhibited at higher rottlerin concentrations of 30-100 µM, but the concentrations of rottlerin used in our assays were too low to affect these kinases. The only other kinase reported to be significantly inhibited by this compound in the low micromolar range is calmodulin kinase III (rottlerin IC50 of 5.3 µM). Therefore, we used a dominant negative PKC-delta mutant, DNPKC-delta (K367R), to discriminate further the role of PKC-delta in PTH effects on IGFBP-5. DNPKC-delta significantly blocked the PTH regulation of IGFBP-5, whereas DNPKC-epsilon had no effect on PTH-(1-34) induction of IGFBP-5. PTH-(3-34) stimulation of IGFBP-5 transcript was also partially inhibited by both rottlerin and DNPKC-delta . The failure of these agents to completely inhibit the PTH-(3-34) effect suggests that a fraction of the PTH-(3-34) regulation of IGFBP-5 transcript is mediated by a pathway that is both PKA and PKC-delta independent. There are a number of signaling pathways that could be stimulated by Gbeta gamma subunits released after PTH-(3-34) stimulation of Gq/11alpha through the PTH receptor (3). The IGFBP-5 gene has been shown to be under the transcriptional control of a plethora of transactivating factors and, subsequently, a large number of signaling pathways (28). Thus beta gamma -subunits released by PTH-(3-34) could potentially mediate the stimulation of a pathway(s) whose activity also regulates IGFBP-5.

Previous attempts to investigate whether the PKC pathway was involved in PTH regulation of IGFBP-5 were performed by attempting to mimic the PTH effect with PMA. Nasu et al. (25) found that PMA was unable to mimic PTH stimulation of IGFBP-5 transcript. We also found that PMA could not induce IGFBP-5 mRNA; furthermore, it inhibited the stimulation of IGFBP-5 by PTH-(1-34). Because PMA selectively activated PKC-alpha in our cells, as evidenced by its translocation to the cell membrane, PKC-alpha appears to have an inhibitory effect on IGFBP-5 transcription. Our demonstration that PMA is also able to partially abrogate the stimulation of IGFBP-5 mRNA induced by forskolin or 8-BrcAMP indicates that the inhibitory effects of PMA occur through cross-regulation of the PKA pathway downstream of adenylyl cyclase. The inhibitory effects of PMA were also directly demonstrated in osteoblast-enriched cultures from fetal rat calvaria, where PMA was shown to inhibit basal expression of IGFBP-5 mRNA (28). PMA could also be regulating IGFBP-5 through the downregulation of PKC-delta ; however, this seems unlikely, given that induction of IGFBP-5 by forskolin and 8-BrcAMP likely occurs by PKA activation independently of PKC-delta regulation.

IGF-I has been shown to act through phosphatidylinositol 3-kinase to regulate IGFBP-5 gene expression in vascular smooth muscle cells (6). Osteoblasts also produce IGF-I, and PTH stimulates its synthesis through a PKA-dependent mechanism. We found that, after 6 h of treatment with exogenously added IGF-I, IGFBP-5 transcript levels were not changed in our cells (M. S. Erclik, unpublished observations), a finding consistent with previous reports by others (4). Thus it is unlikely that there is an IGF-I component to the PTH-induced accumulation of IGFBP-5 mRNA under our assay conditions.

Because there were very low amounts of the transcript in unstimulated cells, it is likely that PTH induces IGFBP-5 mRNA through transcriptional mechanisms. The IGFBP-5 promoter has been cloned and well characterized. A number of AP-2 consensus binding sites have been identified throughout the length of the promoter, and studies in human fibroblasts have demonstrated AP-2 regulation of IGFBP-5 gene expression (5). Because AP-2 has been shown to mediate transcription events in response to both PKA and PKC activation (13), it is an attractive hypothesis to suggest that PTH-stimulated PKC activation works through AP-2 to stimulate IGFBP-5 gene expression. Further studies to characterize the molecular mechanisms of regulation of the IGFBP-5 promoter by PTH are necessary to lend insight into the complex regulation by both PKA and PKC of IGFBP-5 gene expression.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the Canadian Institute of Health Research of Canada.


    FOOTNOTES

Address for reprint requests and other correspondence: J. Mitchell, Dept. of Pharmacology, Univ. of Toronto, 1 King's College Circle, Rm. 4342, Toronto, ON, M5S 1A8 CANADA (E-mail: jane.mitchell{at}utoronto.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00417.2001

Received 18 September 2001; accepted in final form 24 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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