differential regulation of mrnas encoding three protein-tyrosine phosphatases by insulin and...

Vol. 188, No. 3, 1992

No’vember ;S, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1305-1311

Differential Regulation of mRNAs Encoding Three Protein-Tyrosine Phosphatases by Insulin and Activation of Protein Kinase C

Naotake Hashimoto and Barry J. Goldstein’

Research Division, Joslin Diabetes Center and Department of Medicine, Brigham and Women’s Hospital

and Harvard Medical School, Boston, MA 02215

Received September 16, 1992

SUMMARY: Protein-tyrosine phosphatases (PTPases) play an essential role in the control of signalling through phosphotyrosine pathways. Since little is known about the regulation of these enzymes, we examined the effect of insulin and phorbol 12-myristate 13-acetate (PMA) treatment of well-differentiated rat hepatoma (Fao) cells on the expression of mRNAs encoding three major PTPase homologs in liver: PTPaselB, an intracellular enzyme with a single conserved PTPase domain, and two tandem-domain, transmembrane PTPases, known as LAR and LRP. Treatment of serum-deprived cells with 100 nM insulin increased the abundance of the 4.3 kb and 1.6 kb mRNAs encoding PTPaselB on Northern analysis by 1.6 and 3.1 -fold, respectively (p (0.02). Similarly, exposure to 100 rig/ml PMA increased the 4.3 and 1.6 kb PTPaselB mRNAs by 4.5 and 5.7-fold, respectively (~50.035). In contrast, treatment with insulin or PMA had no significant effect of the abundance of mRNA encoding either LAR or LRP. PMA appeared to have a transcriptional effect on the PTPaselB gene by a protein kinase C-mediated mechanism. The increase in PTPaselB mRNA expression by insulin and PMA suggests that this PTPase may provide feed-back regulation of signalling through the insulin action pathway as well as a potential link between the action of protein kinase C and the regulation of specific phosphotyrosine residues in cells. 0 199~ Academic P~,x;, I-)~

Post-translational modification of proteins by phosphorylation on tyrosine residues is

an essential regulatory mechanism for the control of a variety of specialized cellular

functions. Recently, a large family of protein-tyrosine phosphatases (PTPascs; E.C.

3.1.3.48) that reverse the phosphorylation of tyrosyl residues and contribute to the overall

control of signal transduction through these pathways has been characterized by enzyme

purification and molecular cloning (for recent reviews see refs. 1,2). Our laboratory is

particularly interested in PTPases that are involved in the dephosphorylation of

phosphotyrosine residues in the signalling pathway for insulin and other related growth

tTo whom correspondence should be addressed at Division of Endocrinology and Metabolic Diseases, Jefferson Medical College, Room 349, Alumni Hall, 1020 Locust St. Philadelphia, PA 19107-6799.

Vol. 188, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS . e

factors (3). We have recently identified three PTPase homologs that are highly expressed in

liver and muscle and are candidate enzymes for having a physiological role in the regulation

of hormone action in these tissues (46). These enzymes include PTPaselB, which has a

single conserved PTPase domain and is associated with the endoplasmic reticulum (7-lo), and

two PTPases that have a receptor-like transmembrane structure, including the tandem PTPase

domain enzymes LAR (for Leukocyte Antigen Related) and LRP (for LCA-related

phosphatase; also called RPTP-a) (1 l-14).

To gain some initial insight into the potential regulation of these PTPases at the level

of mRNA expression, we examined the effect of insulin and phorbol 12-my&ate 13-acetate

(PMA) on PTPase mRNA expression in well-differentiated rat hepatoma (Fao) cells. The

mRNA for these PTPases was found to be differentially regulated. Messenger RNA for LAR

and LRP was not affected by these agents. In contrast, the mRNAs encoding PTPaselB

mRNA were increased by treatment with insulin or PMA, suggesting a potential mechanism

for feed-back desensitization of signalling through the insulin action pathway as well as a

potential link between the action of protein kinase C and the cellular regulation of specific

phosphotyrosine signals.

METHODS AND MATERIALS

Cell Culture: The well-differentiated rat hepatoma Fao cell line (15) was kindly provided by Dr. C. Ronald Kahn (Joslin Diabetes Center). Fao cells were grown in monolayer using RPMI-1640 medium (GIBCO-BRL, Gaithersburg, MD) containing 10% fetal bovine serum to 80% confluence, washed with phosphate-buffered saline (PBS) and maintained in serum-free medium for 30-40 hr prior to the addition of the indicated experimental agent.

Northern Blot Analysis: After removing the medium, the cell monolayer was washed with PBS and total RNA was extracted by homogenization in buffered 4M guanidinium thiocyanate, extraction with phenol/chloroform and precipitation from isopropanol (16). Electrophoresis of samples containing 20 pg of total RNA was performed in 1% agarose/O.66M formaldehyde gels as described (17). The gels were rinsed twice for 5 min in water, soaked for 30 min in 0.15M NaCl-O.OSM NaOH, rinsed and soaked again for 30 min in 0.15M NaCl-O.lM Tris-HCl, pH 8.0. Capillary transfer to Duralon-UV nylon membranes (Stratagene, La Jolla, CA) was performed with 10X SSC (1X SSC = 0.15M NaCl in 0.015M Na&I!itrate, pH 7.0). After transfer, the RNA was bound to the membrane by UV crosslinking. Prehybridization was performed for 2 hr at 42” C in a solution containing 50% formamide, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), 1M NaCl, and 100 pg/ml denatured salmon sperm DNA. Rat cDNA inserts encoding each of the PTPases were obtained by cDNA amplification or cDNA library screening as described previously (4) and labeled by random hexamer priming with a-[?I-dCTP (18). Hybridization was performed by adding 0.5 to 1 x 106 cpm of probe per ml of prehybridization solution and incubating at 42°C for 20 hr. Filters were washed three times in 2X SSC/O. 1% SDS at room temperature for 15 min each and then in 0.1X SSC/O. 1% SDS

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Vol. 188, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at 55” C for 30 min. The damp filters were then exposed to Kodak X-Omat AR film at with a DuPont Cronex Lightning Plus intensifying screen at -80” C.

Data Analysis: Autoradiograms were quantitated by scanning on a computing densitometer (Molecular Dynamics). Sets of data were compared with two-tailed Student’s t- tests using Instat software (GraphPad, San Diego, CA).

RESULTS

Expression of PTPase mRNAs in rat Fao hepatoma cells: PTPaselB mRNA was

expressed as two transcripts of 4.3 and 1.6 kb, as we have found in normal rat tissues (4) and

as also reported by Guan et al. (7) (Figure 1). The mRNA transcripts for rat LRP and LAR

were expressed in Fao cells as 3.0 kb and 8.0 kb transcripts, respectively, identical to the

major mRNA species for these PTPases in normal rat liver tissue (5)‘. The strength of the

signal from each of the Northern blots using 20 pg of total RNA is a indication of the

relative abundance of mRNA for these PTPases in the hepatoma cells.

Effect of insulin on PTPase mRNA expression: Cells were grown to 80%

confluence and made quiescent by serum starvation for 40h. At that time, insulin was added

at 100 nM, and mRNA was prepared from the cells after an additional 3h of incubation.

Northern blot analysis showed that the expression of both PTPaselB transcripts was increased

significantly by insulin treatment (Figure 1). Quantitation of the Northern blots revealed that

the 4.3 kb mRNA was increased by 1.6-fold (n=6; p=O.O20), and the 1.6 kb mRNA was

increased by a mean of 3. l-fold (n=6; p<O.OlO). In contrast, 3h of insulin treatment had

no detectable effect to increase or decrease the abundance of mRNA for either LAR or LRP.

A time course of the insulin effect on the expression of PTPaselB mRNA revealed that the

mRNA increase was near maximal by 3h of insulin treatment and did not increase further for

either transcript at 6h of incubation (Figure 1) .

Effect of PMA on PTPase mRNA expression: After 3h of treatment with 100

rig/ml PMA, significant increases were observed in the abundance of mRNA for both

PTPaselB transcripts (Figure 2). Quantitation of this effect revealed that the 4.3 kb mRNA

was increased by 4.5-fold over basal (n=5; p=O.O35) and the 1.6 kb mRNA was increased

5.7-fold (n=5; p=O.O30). In contrast, the expression of mRNA for LAR or LRP was

unchanged by incubation with PMA. A time course showed that PMA coordinately affected

the expression of both PTPaselB transcripts which peaked at 3h and gradually returned

‘Zhang, W-R., Hashimoto, N., and Goldstein, B.J., manuscript in preparation.

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PMA 4a-PDD

Time (hrs) ,o 0 3 6 Time (hrs) 0 0.5 1 3 0 1 3

4.3 kb - 4.3 kb -

PTPaselB PTPaselB

1.6 kb - 1.6 kb -

-------

lAR 8.0 kb - LAR 8.0 kb ---)

----_--__ ------- ----_--_

0 1 LRP 3.0 kb - LRP 3.0 kb - 0 2

ml. Effect of insulin on expression of PTPase mRNAs in rat Fao Hepatoma cells. Total RNA was prepared from quiescent control cells and after incubation with 100 nM insulin for the indicated period of time. Twenty microgram samples of RNA were denatured and subjected to electrophoresis in 1% agarose10.66 M formaldehyde gels prior to Northern blot analysis with individual rat cDNA probes for PTPaselB, LAR and LRP as described in Methods. A composite autoradiogram from three separate blots hybridized with the indicated cDNA probe is shown.

Effect of PMA and 4~PDD on expression of PTPase mRNAs in rat Fao Figure 2. Hepatoma cells. Total RNA was prepared from quiescent control cells and after incubation with 100 rig/ml PMA or 4a-PDD for the indicated period of time. Twenty microgram samples of RNA were subjected to Northern blot analysis with cDNA probes for PTPaselB, LAR and LRP as described in the legend to Figure 1. A composite autoradiogram from separate blots hybridized with the indicated cDNA probe is shown.

towards basal levels by 16 to 24 hours. A dose-response for PMA showed that 100 rig/ml

gave a maximal effect on increasing the expression of the two mRNAs encoding PTPase 1B

(data not shown).

We further assessed whether the effect of PMA was mediated by the activation of

protein kinase C by treating quiescent Fao cells with the inactive PMA analogue k-phorbol

12,13-didecanoate (4ar-PDD) at 100 rig/ml for up to 3 hours (Figure 2). This analogue did

not affect the expression of either of the PTPaselB mRNAs or the mRNAs encoding LAB

and LRP. To evaluate whether the effect of PMA on increasing the expression of PTPaselB

mRNAs was mediated at a transcriptional or post-transcriptional level, the half-life of

PTPaselB mRNA was evaluated in PMA-stimulated Fao cells by actinomycin-D treatment

(Figure 3). Each of the two PTPaselB mRNA transcripts exhibited a similar half-life of

-4h in the control cells not pre-treated with PMA. In the PMA-stimulated cells, the half-

life of the two PTPaselB mRNAs was similar and estimated to be - 3h. Therefore, PMA

did not increase the half-life of the PTPaselB mRNA transcripts, suggesting that the effect of

PMA to increase the steady-state level of PTPaselB mBNA is mediated by a direct

transcriptional effect on the PTPaselB gene.

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101 0 1 2 3 4

Time (h) Evaluation of PTPaselB mRNA half-life in Fao cells treated with PMA. Figure 3.

Quiescent Fao cells were incubated with control buffer (--e--) or 100 rig/ml PMA (--P-) for 3 h prior to the addition of actinomycin-D (5 pglml) at time 0 to arrest the synthesis of new RNA. At each subsequent time point, total RNA samples were prepared and Northern blot analysis was performed as described in the legend to Figure 1 using a cDNA probe for PTPaselB. Blot autoradiograms were quantitated by densitometric scanning and the combined level of the two PTPaselB mRNA transcripts was plotted on a semi-logarithmic scale. A regression line is shown for each data set.

DISCUSSION

Over the past few years, our understanding of PTPases as an extensive family of

homologous proteins that influence a variety of cellular signal transduction pathways has

advanced dramatically (1,2). While the precise cellular role of various PTPase homologs is

still uncertain, some insight into their physiology has been provided by investigations into

aspects of their regulation.

Since insulin resistance, particularly at post-binding sites, is a hallmark of human and

several animal models of diabetes, it is of interest that a number of studies have demonstrated

alterations in tissue PTPase activities in the diabetic state. Alterations in tissue particulate

and cytosol PTPase activity towards the insulin receptor have been observed (6,19-22), and

insulin itself has been shown to modulate the activity of PTPases in tissues (6,23). In a

similar fashion, states associated with increased activity of protein kinase C, such as

starvation, are also associated with insulin resistance and reduced insulin receptor tyrosine

kinase activity (24). PMA, which activates protein kinase C, has also been shown to affect

PTPase enzyme activities in cultured cells (25). While the individual PTPase enzymes that

regulate various aspects Of the insulin action pathway are not known, these types of studies

have provided data that PTPase activities in insulin-sensitive tissues may be altered in a way

that can Potentially Play an important role in the pathophysiology of insulin resistance

associated with diabetes mellitus.

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In the present study, we report that insulin increases expression of the mRNA

transcripts encoding PTPaselB in Fao rat hepatoma cells. The time course of the response of

PTPaselB mRNAs to insulin is similar to that reported for the c-myc mRNA response to

insulin in serum-deprived H35 rat hepatoma cells which peaked at 2h (26). These findings

are also of interest in the light of work by Meyerovitch et al. described in ref. (6), which

demonstrated that physiological concentrations of insulin induced an increase in a particulate

fraction PTPase enzyme activity in Fao hepatoma cells. The potential relevance of PTPaselB

to insulin action was demonstrated by studies performed by Cicirelli and Tonks and their

colleagues (27,28), in which a soluble fragment of PTPaselB purified from placenta was

microinjected into Xenopus oocytes and was found to block insulin-stimulated S6 peptide

phosphorylation and retard insulin-induced oocyte maturation. Thus, the induction of

PTPaselB expression by insulin suggests a potential mechanism for feedback desensitization

of phosphotyrosine signalling through the insulin action pathway.

Treatment of serum-deprived cells with PMA also increases the abundance of both

PTPaselB mRNAs. In isolated rat adipocytes, Begum et al. (29) did not find any effect of 1

PM PMA on overall PTPase activity. However, after PMA treatment, Butler et al. (25)

found an increase in activity of a cytosolic 40 kDa PTPase in a soluble fraction of human

erythroleukemia cells, which may represent a catalytic fragment of PTPaselB (30).

Brautigan and Pinault (31) have also shown that a membrane PTPase complex with a catalytic

subunit that reacts with antibodies to PTPaselB sequences can be rapidly activated in CV-1

cells after incubation with 100 nM PMA for 5 min. These studies provide further evidence

for regulation of PTPase activities, possibly involving PTPaselB, at multiple levels that

include both transcriptional and post-translational mechanisms.

In summary, we have found differential regulation of mRNA encoding three abundant

PTPase homologs in well-differentiated hepatoma cells. The mRNAs encoding PTPaselB

appear to be regulatable by a variety of signalling pathways. This may reflect the potential

cellular role of PTPaselB on feed-back regulation of signalling through the insulin action

cascade. In addition, the effect of PMA to increase the expression PTPaselB mRNA

suggests another potential link for “cross-regulation” of pathways involving the action of

protein kinase C and tyrosine dephosphorylation (3 1).

ACKNOWLEDGMENTS

This work was supported by NIH grant ROl-DK43396 to Dr. Goldstein. Molecular and biochemistry core laboratory services were provided by the Joslin Diabetes and Endocrinology Research Center Grant DK36836.

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Vol. 188, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS .

4

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differential regulation of mrnas encoding three protein-tyrosine phosphatases by insulin and...

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