©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tumor Necrosis Factor Facilitates Nuclear Actions of Retinoic Acid to Regulate Expression of the Alkaline Phosphatase Gene in Preosteoblasts (*)

Shehnaaz S. Manji , Hong Zhou , David M. Findlay , T. John Martin , Kong Wah Ng (§)

From the (1) Department of Medicine, The University of Melbourne, St. Vincent's Hospital, Fitzroy, Victoria, 3065, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

This study examines the molecular mechanisms of interaction between tumor necrosis factor (TNF ) and retinoic acid on the expression of the alkaline phosphatase gene by rat clonal preosteoblastic cells. In this cell line, alkaline phosphatase mRNA was not constitutively expressed but was progressively induced by treatment with 1 µ M retinoic acid, detectable by 6 h. Combining retinoic acid with 0.6 n M TNF resulted in alkaline phosphatase mRNA appearing by 2 h, as well as enhanced expression above that observed with retinoic acid alone at 6, 12, and 24 h. Nuclear run-on analysis showed constitutive transcription of the alkaline phosphatase gene in control and TNF -treated cells. At 4 h, retinoic acid, alone or combined with TNF , increased alkaline phosphatase gene transcriptional rate by 2-fold. However, at 24 h, while no retinoic acid effect was retained, retinoic acid plus TNF resulted in a 5-fold increase in alkaline phosphatase transcriptional rate. Examination of the distribution of nuclear alkaline phosphatase mRNA demonstrated that pre-spliced precursor mRNA was localized to the nuclear matrix in control and all treatment groups. Retinoic acid caused a time-dependent accumulation of mature, spliced alkaline phosphatase mRNA located in the non-matrix and cytoplasmic fractions, implying a post-transcriptional action of retinoic acid in nuclear processing and nucleocytoplasmic transport. Adding TNF with retinoic acid greatly enhanced this effect, which was observed after 4 h, prior to any detectable interaction between TNF and retinoic acid on gene transcription. In sharp contrast, only a negligible amount of nuclear processing occurred in control and TNF -treated cells. This study reveals distinct interactions between TNF and retinoic acid at post-transcriptional as well as transcriptional levels to regulate expression of the alkaline phosphatase gene in preosteoblasts.


INTRODUCTION

Tumor necrosis factor (TNF )() is a multifunctional cytokine produced chiefly by infiltrating macrophages and monocytes at sites of inflammation to regulate cell function locally. The actions of TNF are mediated by specific cell surface receptors present on virtually all cells examined. Binding of TNF followed by internalizaton of the cytokine-receptor complex results in the activation of multiple signal transduction pathways, transcription factors, and regulation of transcription of a wide array of genes (for reviews, see Refs. 1-4).

Cytokines characteristically possess multiple functions with considerable overlap in their biological actions (5) . There are many examples of interaction between TNF and other cytokines. For instance, many of its functions are synergistic with interleukin 1. Together, they have pivotal roles in immune and inflammatory responses, mediating host reactions to stimuli as diverse as infections and tumor growth. Some of the mechanisms of cytokine interaction have been studied in detail. Thus, it has been shown that the induction of interleukin 6 by TNF or interleukin 1 required the participation of at least three different cis-acting elements in the promoter region of the interleukin 6 gene, which, in turn, came about through the activation of more than one signaling pathway or transcription factor (6, 7, 8) . TNF also cooperates with systemic factors such as 1,25(OH)-dihydroxyvitamin Dor retinoic acid to induce the differentiation of human leukemia-derived myeloid cell lines (9, 10) , but the manner in which these interactions occur remains largely unexplored.

We have previously reported that TNF potentiated retinoic acid-induced increase in alkaline phosphatase activity in a rat clonal pre-osteoblastic cell line, UMR 201 (11) . Other investigators have also shown that osteoblasts are targeted by TNF . For instance, treatment with TNF decreased collagen synthesis in fetal rat calvariae (12) and stimulated proliferation in human osteosarcoma as well as trabecular bone cells (13, 14) . TNF has also been shown to stimulate osteoclastic bone resorption in organ culture (12) , possibly by stimulating the proliferation of marrow mononuclear cells and enhancing the differentiation of committed osteoclast progenitors (15) . The effects of TNF on mature osteoclasts in vitro are likely to be indirect, requiring the mediation of cytokines secreted by co-cultured osteoblasts (16, 17, 18, 19) .

The object of this study was to examine in detail the manner in which TNF interacted with retinoic acid in the expression of the alkaline phosphatase gene at transcriptional and post-transcriptional levels. We have recently identified an important mechanism of action of retinoic acid whereby the steady state mRNA levels of alkaline phosphatase were increased primarily by enhancement of nuclear processing rather than by an increase in gene transcription (20) . The present results show that TNF regulates alkaline phosphatase gene expression by facilitating the actions of retinoic acid at post-transcriptional as well as transcriptional levels.


EXPERIMENTAL PROCEDURES

Materials

-Modified minimum essential medium (-MEM) was purchased from Flow Laboratories Australia, Pty Ltd. (Mt. Waverley, Victoria, Australia). Fetal bovine serum (FBS) was a product from Grand Island Biological Co. Laboratories (Melbourne, Victoria, Australia). [-P]dCTP, [-P]UTP, and [-P]ATP were purchased from Amersham, Australia, Pty. (Sydney, New South Wales, Australia). All- trans retinoic acid (retinoic acid) was purchased from Sigma. Dr. M. Young (NIDR, National Insitutes of Health) provided the cDNA for chicken actin. Dr G. Rodan (Merck Sharpe and Dohme) provided the cDNA for rat alkaline phosphatase (21) . Recombinant murine TNF was obtained from Dr. R. G. Hammonds, Jr. (Genentech Inc., San Francisco). Nuclease S1 and RNase-free DNase I were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). All other reagents were of analytical grade obtained from standard suppliers.

Cell Cultures

UMR 201 cells were routinely grown in -MEM containing 10% FBS (11) . Incubation was carried out at 37 °C in a humidified atmosphere equilibrated with 5% COin air.

Northern Blot Analysis

Total RNA was isolated with guanidine thiocyanate (23) , separated in a 1.5% agarose-formaldehyde gel, and transferred to nylon filters (24) . Filters (Hybond-N, Amersham, UK) were hybridized overnight in SSPE buffer (1 SSPE = 150 m M NaCl, 10 m M NaHP0H0, 1 m M sodium EDTA, pH 7.4) as previously described (20) . cDNA probes were nick-translated with [-P]dCTP to a specific activity of 1 10dpm/µg of DNA (Boehringer Mannheim). The filters were washed sequentially in 2 SSPE with 0.1% SDS at 42 °C for 15 min, 1 SSPE with 0.1% SDS at 65 °C for 30 min, and finally 0.1 SSPE with 0.1% SDS at room temperature for 15 min. Specifically bound probe was visualized by autoradiography and quantified by densitometry at appropriate exposures (Molecular Dynamics, model 300A). Relative mRNA levels were normalized for loading variability by comparison with actin mRNA levels in the same filters.

Nuclear Run-on Analysis

Transcriptional rates of alkaline phosphatase and actin genes were determined by nuclear run-on analysis as previously described (25) . UMR 201 cells were grown in 500 cmtrays in -MEM containing 10% FBS until 80% confluent before the medium was changed to -MEM containing 2% vitamin A-deficient FBS, and treatment commenced with 1 µ M retinoic acid, 0.6 n M TNF , or 1 µ M retinoic acid together with 0.6 n M TNF for 4 and 24 h, respectively. The cDNA for alkaline phosphatase (10 µg) and actin (5 µg) were denatured and immobilized on nitrocellulose (Hybond-C, Amersham, UK). Quantitation was carried out by densitometry of submaximal signals and normalized with reference to the actin signal on the same filter.

Subcellular Fractionation

Subcellular fractions of UMR 201 cells, consisting of nuclear matrix with which precursor mRNA is almost exclusively associated, pooled DNase I/salt eluate enriched in mature mRNA, nuclear membrane, and cytoplasm were obtained as previously described (20) using a method originally described by Leppard and Shenk (26) . UMR 201 cells were grown in 500-cmtrays in -MEM containing 10% FBS until 80% confluent before treatment with 1 µ M retinoic acid, 0.6 n M TNF , or 1 µ M retinoic acid together with 0.6 n M TNF in -MEM containing 2% vitamin A-deficient FBS for the indicated incubation times.

Total RNA from the subfractions was prepared using the method described by Gough (27) . Supernatant from the various RNA-containing subfractions was added to 4 ml of protein-denaturing buffer containing 7 M urea, 350 m M NaCl, 10 m M EDTA, 1% SDS, and 10 m M Tris-HCl, pH 7.4. RNA was extracted by adding 8 ml of phenol/chloroform/isoamylalcohol (50:50:1) and precipitated with 8 ml of isopropanol. The RNA pellet was washed once with 70% ethanol and resuspended in TE buffer (10 m M Tris-HCl, pH 8.0, 1 m M EDTA, pH 8.0) at a concentration of 2 µg/µl. For Northern blot analysis, total RNA was separated in a 1.5% agarose-formaldehyde gel as described above. Equal loading was confirmed by staining the gels with ethidium bromide.

S1 Nuclease Protection Assay

S1 nuclease protection assay was performed as previously described (20, 28, 29) . A 27-nucleotide (5`-CAATATAGCTGCCACATGCCTGCTCAC-3`) complementary to the start of the second intron of the rat liver alkaline phosphatase gene (30) was labeled with [-P]ATP at the 5`-end. 50,000 cpm of the labeled nucleotide was hybridized to 20 µg of total RNA from each of the subcellular fractions obtained as described above. The protected DNA-RNA hybrid was extracted with phenol-chloroform, ethanol precipitated, and electrophoresed on a 20% acrylamide gel containing 8 M urea; the dried gel was then subjected to autoradiography.

Polymerase Chain Reaction Amplification of Reverse-transcribed mRNA

First strand cDNA was synthesized from 2 µg of total RNA by incubating for 1 h at 42 °C with 15 units of AMV reverse transcriptase (Promega) following oligo(dT) priming. 2 µl of this reaction mixture was submitted to PCR to amplify the region of the rat alkaline phosphatase gene transcript across exons 4 and 5 to include intron 4 (see Fig. 5 A). Primers were a 5`-primer (5`-AGAAAGAGAAAGACCCCAGTT-3`) representing nucleotides 1-21 of exon 4 and a 3`-primer (5`-CTTGGAGAGAGCCACAAAGG-3`) representing nucleotides 97-116 of exon 5 (30) . 20 cycles of amplification were sufficient to yield a PCR product corresponding to spliced mRNA, whereas 30 cycles of amplification were required to demonstrate product corresponding to intron-containing (unspliced) mRNA. Amplification using Taq DNA polymerase (Boehringer Mannheim) with an annealing temperature of 55 °C was employed in a Perkin-Elmer Corp. 480 thermal cycler. 20 µl of each PCR reaction mixture was run on a 2% agarose gel and transferred to nylon; products were authenticated by probing with a mixture of 3 digoxigenin-labeled oligonucleotides corresponding to sequences within exons 4 and 5 as well as in intron 4 (see Fig. 5A). Oligonucleotides were labeled with digoxigenin-dUTP using a 3`-tailing kit (Boehringer Mannheim). Hybridization was carried out with 2 pmol/ml labeled oligonucleotide in a buffer containing 5 SSC, 0.02% SDS, 0.1% sarcosine, and 100 µg/ml poly(A) at 55 °C for 14 h. Detection was by chemiluminescence using Lumigen PPD (Boehringer Mannheim) according to the manufacturer's instructions. Products of the expected sizes were obtained, viz. 636 bp for unspliced and 236 bp for spliced species.


Figure 5: Subcellular localization of prespliced and mature alkaline phosphatase mRNA in UMR 201 cells determined by reverse transcriptase-PCR analysis. A, the alkaline phosphatase gene comprises 13 exons (30). The locations of the oligonucleotides used for reverse transcriptase-PCR primers are indicated by the arrows; oligonucleotide probes to authenticate PCR products are indicated by solid bars and labeled 1-3. Oligonucleotide 4 was used for S1 nuclease analysis. Intron 4 is 400 bp. Splicing out intron 4 results in a PCR product of 236 bp; unspliced mRNA yields a reverse transcriptase-PCR product of 636 bp. B, subcellular fractions were prepared from control cells or cells treated for 8 h with 0.6 n M TNF ( T), 10 M retinoic acid ( R), or TNF combined with retinoic acid ( RT). C represents control (untreated) cells. Fractions are as follows: Cyto, cytoplasmic; Memb, nuclear membrane; DNase I, DNase I/high salt eluate; and NM, nuclear matrix. 30 cycles of amplification of reverse-transcribed mRNA from each fraction gave rise to an amplified fragment of approximately 636 bp only in the nuclear matrix fraction. The fragment hybridized to specific digoxigenin-labeled oligonucleotides ( 1-3) complementary to exons 4 and 5 and intron 4. Chemiluminescence was detected by exposure of filters to x-ray films at room temperature. The amplification reaction, which produced a 236-bp product corresponding to exon 4 spliced to exon 5, was carried out for 20 cycles. Reverse-transcribed mRNA was also amplified for 20 cycles using rat GAPDH primers to produce the expected 414-bp fragment, which hybridized to a specific digoxigenin-labeled internal oligonucleotide (see ``Experimental Procedures'').



To ensure equal starting quantities of RNA in each sample, the reverse-transcribed material was also amplified using oligonucleotide primers specific for rat GAPDH (31) . A 414-bp fragment was amplified using as 5`-specific olignucleotide, GAPDH-4 (5`-CATGGAGAAGGCTGGGGCTC-3`, representing nucleotides 306-325 of rat GAPDH) and 3`-specific oligonucleotide, GAPDH-5 (5`-AACGGATACATTGGGGGTAG-3`, representing nucleotides 701-720). Products were verified with a digoxigenin-labeled internal sense strand oligonucleotide, GAPDH-1 (5`-GCTGTGGGCAAGGTCATCCC-3`, representing nucleotides 640-659) using the hybridization conditions described above.


RESULTS

Alkaline Phosphatase mRNA Steady State Levels

We have previously reported that alkaline phosphatase activity was highly induced in UMR 201 cells by retinoic acid with a peak effect observed at 1 µ M (32) . Although TNF was without effect in inducing alkaline phosphatase activity, retinoic acid-induced alkaline phosphatase activity was greatly enhanced when UMR 201 cells were treated with TNF and retinoic acid together. This effect was dose dependent with respect to TNF , plateauing after 0.06 n M (33) . In the present study, UMR 201 cells were treated with a maximal concentration of 0.6 n M TNF , 1 µ M retinoic acid, or retinoic acid together with TNF . Total RNA prepared from the cells after treatment for 2, 6, 12, or 24 h, respectively, was separated in a 1.5% formaldehyde-agarose gel, transferred to nylon filters, and probed for alkaline phosphatase mRNA in a Northern blot analysis. The results show that retinoic acid progressively stimulated the expression of alkaline phosphatase mRNA, detectable by 6 h (Fig. 1). Combined treatment with retinoic acid and TNF resulted in much earlier expression of alkaline phosphatase mRNA, now detectable at 2 h. In addition, an increase in the steady state level of alkaline phosphatase mRNA, above that obtained with retinoic acid treatment alone, was observed at all other time points. mRNA for alkaline phosphatase was not expressed in control (untreated) UMR 201 cells, and TNF alone had no effect on alkaline phosphatase mRNA expression. These results correlate very well with our previous report on the action of TNF and retinoic acid on alkaline phosphatase enzymatic activity in these cells (33) .

To achieve a clearer understanding of the mechanisms of action of TNF and the manner in which it interacts with retinoic acid, the individual and combined effects of these factors on the transcriptional and post-transcriptional regulation of expression of the alkaline phosphatase gene were examined.

Transcription of Alkaline Phosphatase Gene

Transcription of the alkaline phosphatase gene was examined by nuclear run-on analysis after UMR 201 cells were treated, as described above, for 4 or 24 h, respectively (Fig. 2). The first time point of 4 h was chosen to precede the earliest detectable increase in alkaline phosphatase mRNA induced by retinoic acid (6 h, Fig. 1 ). The alkaline phosphatase gene was found to be constitutively transcribed by untreated (control) UMR 201 cells and those treated with TNF alone, despite the absence of detectable alkaline phosphatase mRNA (Fig. 1). After 4 h, there was a small, approximately 2-fold increase in the transcriptional rate of the alkaline phosphatase gene in cells treated with 1 µ M retinoic acid alone. There was also an increase in the rate of transcription in cells treated with retinoic acid and 0.6 n M TNF but only to a similar extent as that induced by retinoic acid alone. After treatment for 24 h, the significant feature was the striking 5-fold increase in the rate of transcription of the alkaline phosphatase gene in cells treated with retinoic acid and TNF (Fig. 2, bottom row). The earlier effect of retinoic acid alone in stimulating alkaline phosphatase gene transcription was transient and no longer observed at 24 h, as previously reported (20) .


Figure 1: Detection of mRNA for alkaline phosphatase ( ALP) mRNA in UMR 201 in a Northern blot analysis. C denotes control (untreated) cells. Cells were treated with 0.6 n M TNF ( T), 1 µ M retinoic acid ( R), or 1 µ M retinoic acid combined with 0.6 n M TNF ( RT) for the indicated times. Total RNA (20 µg) was loaded into each lane. The filter was initially probed with a plasmid containing a 2.4-kilobase cDNA for rat alkaline phosphatase, washed, and reprobed with a chicken actin cDNA for normalization. The arrows point to the positions of the 28 and 18 S ribosomal bands, respectively. This is a representative experiment performed three times with similar results.




Figure 2: Nuclear run-on analysis of transcriptional rates of the alkaline phosphatase and actin genes in UMR 201 cells. Cells were grown in 500-cmtrays either untreated ( C) or treated with 0.6 n M TNF ( T), 1 µ M retinoic acid ( R), or 1 µ M retinoic acid combined with 0.6 n M TNF ( RT) for 4 h ( top row) and 24 h ( bottom row), respectively. Nuclei were prepared, and run-on transcription was allowed to proceed in the presence of 100 µCi of [-P]UTP as described under ``Experimental Procedures.'' Newly synthesized labeled RNA was hybridized to immobilized cDNAs as indicated. ALP, alkaline phosphatase; pBS is the empty vector used to provide a signal corresponding to nonspecific hybridization. Quantitation was carried out by densitometry at appropriate exposures and normalized by reference to the actin signal. This experiment was performed twice, in duplicate, and a representative result is shown here.



Nuclear Processing of Alkaline Phosphatase mRNA

Clearly, there is a discrepancy between the small increase in transcriptional rate of the alkaline phosphatase gene, as well as the time course of those increases in response to the various treatments, and the observed changes in the steady state levels of the alkaline phosphatase mRNA in the Northern blot analysis shown in Fig. 1. To examine post-transcriptional events, therefore, UMR 201 cells were subfractionated to isolate the nuclear matrix, DNase I/salt eluate, nuclear membrane, and cytoplasmic fractions. The extent of nuclear processing is indicated by the relative distribution of precursor and mature alkaline phosphatase mRNA between the subnuclear and cytoplasmic fractions.

UMR 201 cells were treated for 4, 12, and 24 h with 0.6 n M TNF , 1 µ M retinoic acid, or TNF combined with retinoic acid, respectively, and compared with untreated cells. The cells were subfractionated, and total RNA from the various fractions was analyzed in two ways. First, to detect pre-spliced precursor mRNA, solution hybridization to a 27-nucleotide oligomer, complementary to the start of the second intron of the rat alkaline phosphatase gene, was carried out before S1 nuclease digestion of single-stranded nucleic acid was performed (Fig. 3). The protected DNA-RNA hybrid was only detected in the nuclear matrix fraction of control and treated cells, confirming that precursor alkaline phosphatase mRNA was present specifically in that fraction. Although the band in the nuclear matrix fraction of control cells was only faintly visible, it was consistently present in repeated experiments. Fig. 3 shows the results from an experiment where cells were treated for 4 h, but similar results were obtained after 24 h (data not shown).


Figure 3: Subcellular localization of precursor mRNA for alkaline phosphatase in UMR 201 cells using an intronic probe in a S1 nuclease protection assay. Cells were grown in 500-cmtrays untreated (control, C), treated with 0.6 n M TNF , 1 µ M retinoic acid ( R), or 1 µ M retinoic acid combined with 0.6 n M TNF ( RT) for 4 h. The isolated subcellular fractions, obtained as described under ``Experimental Procedures,'' were cytoplasm, nuclear membrane, DNase I/salt eluate, and nuclear matrix. Total RNA was prepared from each fraction, and 20 µg were allowed to hybridize in solution with a 27-nucleotide oligomer, complementary to a sequence in the second intron of the alkaline phosphatase gene, as described under ``Experimental Procedures.'' 50,000 cpm of labeled oligomer was added to each reaction. The protected DNA-RNA hybrid was electrophoresed on a 20% acrylamide gel containing 8 M urea, and the dried gel was subjected to autoradiography. This experiment was performed three times, and a representative result is shown here.



The second analysis was designed to determine whether the precursor mRNA was being processed into mature mRNA, which can be translocated from the nucleus into the cytoplasm. Total RNA from the various subcellular fractions was separated in a 1.5% formaldehyde-agarose gel, transferred to nylon filters, and probed with a 2.4-kilobase full-length cDNA encoding rat alkaline phosphatase (Fig. 4). In contrast to the results obtained in Fig. 3, there was only a minute amount of mature, spliced alkaline phosphatase mRNA in the nuclear membrane fraction of control and TNF -treated cells, despite the presence of precursor mRNA in the nuclear matrix fraction. On the other hand, retinoic acid treatment resulted in the accumulation of mature alkaline phosphatase mRNA mainly within the non-matrix and cytoplasmic fractions, as previously reported (20) . When the cells were treated with the combination of TNF and retinoic acid, not only was there a considerable increase in the amount of mature alkaline phosphatase mRNA located in the subcellular fractions, but it occurred to such an extent that it was present even in the nuclear matrix fraction. This enhancement of nuclear processing of alkaline phosphatase mRNA transcripts with the combined treatment was evident at all time points, beginning at 4 h. This early time point clearly preceded the effect of the combined treatment on increasing the rate of alkaline phosphatase gene transcription beyond that induced by retinoic acid treatment alone (Fig. 2). Importantly, TNF alone did not have any notable influence on nuclear processing.

Although we have previously reported that co-treatment with cycloheximide significantly inhibited the effect of retinoic acid on nuclear processing (20) , a similar experiment could not be performed in this study because the combination of cycloheximide and TNF was toxic to the cells.

Reverse Transcriptase-Polymerase Chain Reaction Analyses

To verify these results and at the same time to seek evidence of elongation of the nascent transcript, we employed reverse transcriptase-PCR to amplify a sequence corresponding to intron 4 of the alkaline phosphatase gene, using primers complementary to exons 4 and 5 (see Fig. 5 A). UMR 201 cells were subfractionated after treatment with TNF , retinoic acid, or TNF combined with retinoic acid for 8 h. Consistent with the S1 nuclease results, a 636-base pair amplified product corresponding to RNA containing intron 4 was present in the nuclear matrix fractions only of control and treated cells (Fig. 5 B). In addition, a 236-base pair product corresponding to spliced mRNA was seen in cells treated with retinoic acid alone or when combined with TNF . There was clearly a much greater abundance of spliced alkaline phosphatase mRNA in all of the subcellular fractions in response to the combined treatment (Fig. 5 B). DNase treatment during the nuclear fractionation procedure ensured that this product did not result from contaminating genomic DNA.


DISCUSSION

We have used a non-transformed clonal rat cell line, UMR 201, with features characteristic of pre-osteoblasts in our studies of osteoblast differentiation. Retinoic acid significantly stimulates the expression of mRNA for alkaline phosphatase, osteopontin, osteonectin, matrix Gla protein, and type I collagen in these cells (22, 32, 33) . Previous studies have shown that retinoic acid and TNF acted in concert to control the level of alkaline phosphatase mRNA in UMR 201 cells (11) . The present results advance our understanding of this interaction. TNF itself had no effect on the transcriptional rate of the alkaline phosphatase gene. When combined with retinoic acid, there was a significant increase in transcriptional rate of this gene over and above the increase caused by retinoic acid alone, but this was clearly evident only when the cells had been treated for 24 h. The Northern blot analysis, however, showed that the increase in steady state level of alkaline phosphatase mRNA induced by the combination of TNF and retinoic acid was demonstrable after treatment for only 2 h, which significantly preceded the induction of alkaline phosphatase mRNA by retinoic acid treatment alone. These results point to the need to consider a potential role for post-transcriptional regulatory events in determining the steady state level of cytoplasmic alkaline phosphatase mRNA.

The alkaline phosphatase gene is constitutively transcribed in control and TNF -treated UMR 201 cells, and this was verified by the demonstration of precursor alkaline phosphatase mRNA in the nuclear matrix fraction of all treatment groups using the sensitive S1 nuclease protection assay as well as reverse transcriptase-PCR. The detection of only minute amounts of mature alkaline phosphatase mRNA in the nuclear membrane fraction of control and TNF -treated cells testifies to the inefficiency of nuclear processing in cells grown under those conditions. It further implies that newly transcribed precursor alkaline phosphatase mRNA must be rapidly degraded within the nuclear matrix fraction.

We have previously described a novel action of retinoic acid, in that it greatly increases the proficiency of nuclear processing of newly transcribed alkaline phosphatase mRNA, enabling the accumulation of mature mRNA in the non-matrix nuclear fractions prior to translocation into the cytoplasm (20) . The mechanism of action of retinoic acid in enhancing nuclear processing is unclear. Factors that will need to be considered include the role of RNA binding proteins and their participation in regulation of the splicing mechanism, stability of native and spliced RNA transcripts, as well as translocation of the spliced product from the nucleus to the cytoplasm. In contrast to our findings, another group has shown that in human peripheral blood mononuclear cells, retinoic acid induced interleukin-1 and interleukin-1 precursor mRNA, but these were not processed into mature mRNA, requiring treatment with cycloheximide to facilitate nuclear processing (34) . These recent findings may imply that the effect of retinoic acid on nuclear processing might be cell-type and/or gene specific. An additional implication of those results is that retinoic acid requires collaboration with additional cofactor(s) to direct or enable intranuclear processing of transcripts. In the present case, TNF serves as one such cofactor to greatly enhance the retinoic acid-induced nuclear processing.

Although TNF alone appeared not to influence either the transcription of the alkaline phosphatase gene or the nuclear processing of the mRNA, it significantly enhanced the post-transcriptional actions of retinoic acid early in the course of the interaction and, later on, also with the transcriptional events. Although it must still be suggested that the enhancement of retinoic acid-induced nuclear processing by TNF may be a consequence of the greatly increased transcriptional rate of the alkaline phosphatase gene induced by the combined treatment, the considerable delay before this became evident is not consistent with this possibility.

A locally secreted cytokine such as TNF or transforming growth factor , acting as an autocrine or paracrine regulator of cell function, is ideally located to modulate the effects of systemic agents. It is interesting to note that the mechanisms of action of TNF in the regulation of alkaline phosphatase gene expression shown in this study contrasted significantly with that of transforming growth factor (20) . Unlike TNF , transforming growth factor increased the transcriptional rate of alkaline phosphatase gene, but that action was not accompanied by increased nuclear processing of precursor mRNA.

In conclusion, this study has revealed that TNF interacts with retinoic acid at post-transcriptional as well as transcriptional levels to regulate alkaline phosphatase gene expression. Since alkaline phosphatase expression is a marker of differentiation in osteoblasts, the enhancement of retinoic acid action by TNF suggests that this cytokine may further promote the differentiation of preosteoblastic UMR 201 cells. Furthermore, this system will be an excellent model in which to study the mechanisms involved.


FOOTNOTES

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

§
To whom correspondence should be addressed. Fax: 613-288-2581.

The abbreviations used are: TNF , tumor necrosis factor ; FBS, fetal bovine serum; PCR, polymerase chain reaction; bp, base pair(s); -MEM, -modified minimum essential medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


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