©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
28-kDa Mammalian Heat Shock Protein, a Novel Substrate of a Growth Regulatory Protease Involved in Differentiation of Human Leukemia Cells (*)

(Received for publication, October 12, 1994; and in revised form, November 17, 1994)

Neil L. Spector (1)(§) Lys Hardy (1) Colleen Ryan (2) Wilson H. Miller Jr. (3) John L. Humes (4) Lee M. Nadler (2) Edward Luedke (5)

From the  (1)Division of Hematology-Oncology, University of Miami School of Medicine, Miami, Florida 33136, (2)Division of Tumor Immunology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, (3)Lady Davis Institute of the Jewish General Hospital, Montreal 4365, Canada, (4)Biochemical and Molecular Pathology, Merck & Co., Rahway, New Jersey 07065, and (5)Amgen Corp., Thousand Oaks, California 91320

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Because of their differentiating effects in neoplastic cells in vitro, the use of retinoids in the treatment of various malignant and premalignant conditions is under investigation. To date, signal transduction pathways involved in retinoid-induced differentiation remain poorly understood. Differentiation of HL-60 cells by all-trans-retinoic acid (tRA) is directly mediated by down-regulation of the serine protease myeloblastin (mbn). In this report, we investigate the possibility that the 28-kDa heat shock protein (hsp28), previously linked to differentiation of normal and neoplastic cells including HL-60, may be regulated by mbn. Using NB4 promyelocytic leukemic cells as a differentiative model, we show that tRA induces initial suppression and subsequent up-regulation of hsp28 protein, mirroring tRA-induced changes in mbn protein. The progressive reduction in hsp28 mRNA levels in response to tRA suggests that changes in hsp28 protein levels might be posttranscriptionally mediated, raising the possibility that hsp28 may be targeted by mbn. To address this, we developed an assay using purified mbn and recombinant hsp28 and now show that hsp28 is hydrolyzed by mbn but not its homologue, human neutrophil elastase. Moreover, mbn does not indiscriminately hydrolyze other proteins. Identifying hsp28 as a substrate of mbn strongly suggests that hsp28 may be a key component of the tRA signaling pathway involved in regulating cell differentiation.


INTRODUCTION

Vitamin A and its retinoid derivatives exert marked biological effects in a variety of normal and neoplastic cells. For example, RA (^1)induces human myeloid leukemic cells to exit the cell cycle and terminally differentiate into mature granulocytes, both in vitro(1, 2) and in vivo(3, 4, 5) . In this context, a novel serine protease, myeloblastin, has been shown to be directly involved in RA-induced differentiation of human HL-60 myeloid leukemic cells(6) . Constitutively expressed in proliferating HL-60 cells, mbn is rapidly down-regulated in response to RA(6, 7) . Inhibition of mbn protein expression appears to correlate with the cessation of cell growth. To directly link mbn down-regulation to changes in cell growth and differentiation, mbn protein expression was specifically inhibited in HL-60 cells using antisense oligonucleotides. This manipulation induced spontaneous growth arrest and differentiation, directly implicating mbn as a key component of the signaling pathway involved in transducing the biological effects of RA (6) . To elucidate on this pathway, we sought to identify proteolytic targets of mbn.

In addition to their role in the development of thermotolerance, members of the highly conserved heat shock protein family have been linked to cell growth and differentiation(8) . Targeting of the 28-kDa mammalian heat shock protein by mitogens(9, 10) , growth regulatory cytokines(11, 12, 13) , and differentiating agents(14, 15, 16, 17, 18, 19) implicated hsp28 as a potential component of signal transduction pathways in both normal and neoplastic cells. Several lines of evidence made it tempting to speculate that hsp28 might be a substrate of mbn. First, hsp28 mRNA was markedly down-regulated by RA during differentiation of HL-60 cells whereas hsp28 protein levels actually increased coincident with the onset of growth arrest(16) . This finding suggested that changes in hsp28 protein levels might be posttranscriptionally regulated. Second, mbn and hsp28 proteins appear to be reciprocally regulated during granulocytic differentiation of HL-60 cells. Third, amino acid sequence analysis of hsp28 revealed several potential serine protease target sites(20) . In the current study, we establish hsp28 as the first reported biological substrate of mbn. Considering hsp28 is a molecular chaperone (21) and key actin binding protein(22) , its regulation by mbn sheds light on potential pathways involved in transducing the differentiative effects of RA in human myeloid malignancies.


EXPERIMENTAL PROCEDURES

Materials

[methyl-^3H]Thymidine (37 kBq) was purchased from ICN Biochemicals. [alpha-P]dCTP (3000 Ci/mmol) was obtained from DuPont NEN. Recombinant hsp28 (rhsp28), alpha-hsp28 mAb, and hsp28 cDNA probe were from StressGene Corp. Rabbit alpha-hsp28 antisera were obtained from Dr. A. P. Arrigo (Lyon, France)(11) . All-trans-retinoic acid and Ponceau S were purchased from Sigma. Purified human neutrophil elastase was from Athens Research and Technology. The ECL immunoblot detection kit was purchased from Amersham Corp. Purified human IgG, goat anti-human horseradish peroxidase-conjugated secondary antibody, and alkaline phosphatase immunoblotting reagents were obtained from Calbiochem. Cell culture reagents were obtained from Life Technologies, Inc. Protein A-Sepharose CL-4B was obtained from Pharmacia Biotech Inc.

Cell Culture

NB4 cells were kindly provided by Dr. M. Lanotte (INSERM, France). Cells were maintained in log growth in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, and 5 mg/ml gentamycin.

Purification of Mbn

Mbn was prepared from human polymorphonuclear cells by modifications of the procedure described by Kao et al.(23) . Briefly, blood from patients diagnosed with chronic myelogenous leukemia was isolated by dextran sedimentation and Ficoll-Hypaque centrifugation, and contaminating red blood cells were removed from the resulting supernatant by hypotonic lysis using NH(4)Cl (0.87%). Leukocytes were obtained by centrifugation (400 times g for 10 min) and resuspended in Hanks' buffered salt solution. Leukocytes were homogenized in 0.34 M sucrose at 4 °C, and following centrifugation, the granules were extracted by sonic disruption in 20 mM HCl. After centrifugation (100,000 times g for 60 min), the supernatant was chromatographed on a Dyematrix Orange A-agarose dye affinity column. Proteins bound to the column were eluted using a NaCl gradient (0.1-2.5 M) in citrate phosphate buffer. Enzymatically active fractions eluted from the column were combined together and further purified on a Trasylol-Sepharose column to remove potential contaminating neutrophil elastase and cathepsin G and then subjected to cation exchange chromatography using a Mono-S HR 5/5 column with the Pharmacia fast protein liquid chromatography system. The resulting material was homogeneous as evaluated by LC electrospray-mass spectrometry, N-terminal amino acid sequence determination, and SDS-PAGE. In addition, no neutrophil elastase or cathepsin G was identified in the final product using immunoblotting and enzymatic assays for these proteases.

In Vitro Proteolysis Assay

rhsp28, dissolved in phosphate-buffered saline + 0.075% Tween 20, and purified mbn were co-incubated at an enzyme:substrate ratio of 1:200-1:500 (mol/mol) in a shaking 37 °C water bath for 12-18 h. The reaction was terminated by boiling in 2 times Laemmli sample buffer and then separated by 12% SDS-PAGE. Purified human neutrophil elastase was co-incubated with rhsp28 under identical conditions to that described above.

Preparation of RNA and Northern Blot Analysis

Total cellular RNA was purified by a modification of the guanidinium thiocyanate/CsCl method as described previously(14) . Intactness and equal loading of RNA were verified by ethidium bromide staining. Equal amounts of RNA were run on a 1.3% agarose gel and transferred by Northern blotting onto a Zeta-Probe membrane (Bio-Rad). Hybridization was performed using a P-labeled cDNA probe from the human hsp28 gene.

SDS-PAGE and Immunoblot Analysis

NB4 cells (5 times 10^6 cells/time point) were lysed in buffer containing 0.5% (v/v) Nonidet P-40, 20 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and aprotinin. Protein concentrations were determined by a modification of the Bradford method, and equal amounts of protein (50 µg) were resolved by 12% SDS-PAGE under reducing conditions. Proteins were transferred to a nitrocellulose filter, and efficiency and equal loading of proteins were evaluated by Ponceau S staining. The filter was blocked for 1 h in TBS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 5% (w/v) lowfat milk. Blots were then hybridized with alpha-hsp28 mAb at 1:1500 final dilution in TBS + 1% (w/v) lowfat milk for 1 h at room temperature. Blots were washed 3 times in TBST (TBS containing 0.2% Tween 20) and then developed using either a goat alpha-mouse alkaline phosphatase-conjugated secondary antibody (1:5000 dilution in TBST) or a goat alpha-mouse horseradish peroxidase-conjugated secondary antibody (1:5000 dilution in TBST). Alkaline phosphatase blots were visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrates, and horseradish peroxidase secondary antibodies were visualized by the ECL detection method.

DNA Determinations

NB4 cells were added to microculture plates at 10^5 cells/well in a final volume of 200 µl. Cell proliferation was determined by pulsing cells with 1 µCi/well [^3H]thymidine for 4 h. Cells were harvested onto glass fiber filters, and filter-bound [^3H]thymidine was counted on a Packard Tri-carb scintillation counter.


RESULTS AND DISCUSSION

In order to establish a link between hsp28 and mbn during RA-induced cell differentiation, we examined the regulation of hsp28 protein in the NB4 acute promyelocytic leukemia (APL) cell line. APL cells remain one of the prototypic models with which to study cellular and molecular differentiative events since these cells undergo granulocytic differentiation in response to RA(1, 2, 3, 4, 5) . As the only established APL cell line, NB4 cells contain the t(15;17)(q22;q12-21) cytogenetic marker, which is uniquely associated with APL and thought to confer RA sensitivity through the expression of a hybrid retinoic acid receptor-alpha protein(24, 25, 26, 27, 28) . Decreased mbn protein levels appear to correlate with RA-induced growth arrest of NB4 cells(7) . However, in contrast to HL-60 cells where mbn is rapidly down-regulated, mbn is initially induced in RA-treated NB4 cells and not inhibited until 96 h, at which time NB4 cells growth arrest(7) . hsp28 protein levels were examined at various time points prior to and following treatment of NB4 cells with tRA (1 µM). During the first 72 h, levels of hsp28 protein progressively decreased, during which time [^3H]thymidine incorporation remained unchanged from that of untreated cells (Fig. 1). After nadiring at 72 h, hsp28 protein levels increased at 96 h with continued induction above base-line levels at 4 and 6 days (Fig. 1). Increased hsp28 protein appeared to correlate with cessation of cell proliferation and 55 and 82% inhibition of [^3H]thymidine incorporation at 4 and 6 days, respectively (Fig. 1). Thus, the initial down- and subsequent up-regulation of hsp28 mirrors that previously described for mbn, where mbn is up-regulated during the initial 24-72 h following tRA and not down-regulated until 96 h(7) .


Figure 1: Increased hsp28 marks tRA-induced growth arrest of NB4 cells. Exponentially growing NB4 cells were cultured in the presence of tRA (1 µM). At the indicated times prior to and following treatment with tRA, equal numbers of NB4 cells were lysed in buffer containing 0.5% Nonidet P-40. Protein concentrations in cell lysates were determined by a modification of the Bradford method(50) . Equal amounts of protein (50 µg/lane) were separated by 12% SDS-PAGE and transferred to a nitrocellulose filter. hsp28 was immunoblotted using an alpha-hsp28 mAb (1:1500 dilution) and visualized with a goat alpha-mouse horseradish peroxidase-conjugated secondary antibody (1:5000 dilution). The blot was developed using the ECL method of detection. The position of hsp28 is indicated. Corresponding cell proliferation was determined by pulsing 10^5 cells/well with 1 µCi of [^3H]thymidine (Thym.) for 4 h and then measuring radioactive incorporation into DNA. The percent control = counts/min (indicated time point)/counts/min (day 0).



Relatively little is known about the mechanism regulating hsp28 protein levels during cell differentiation. To shed light on this process, we examined the expression of hsp28 mRNA during RA-induced differentiation of NB4 cells. Total cellular RNA was isolated at various time points prior to and following treatment with tRA. The predicted 0.9-kilobase size hsp28 transcript was markedly down-regulated between 2 and 6 days following treatment with tRA (Fig. 2A). Intactness and equal loading of RNA were verified by ethidium bromide staining (Fig. 2B) and cross-hybridization to the 28 S ribosomal band (Fig. 2A).


Figure 2: Down-regulation of hsp28 mRNA during differentiation of NB4 cells. NB4 cells were either cultured in control media alone (0 and 48 h) or in the presence of tRA (1 µM) for the indicated periods of time. Total cellular RNA was isolated from cells at each time point using a modification of the guanidinium thiocyanate/CsCl method. Equal amounts of RNA (5 µg/lane) were separated by 1.3% agarose gel electrophoresis and transferred by Northern blotting to a nitrocellulose membrane and then hybridized with a P-labeled cDNA probe cloned from the hsp28 gene. As shown, the probe hybridizes to a 0.9-kDa size transcript (panelA). Cross-hybridization to the 28 S ribosomal band suggests comparable loading of intact RNA. A photograph of the ethidium bromide-stained 28 S ribosomal band is shown as a further control of intactness and equal loading of RNA (panelB). The positions of the 28 S and 18 S ribosomal bands are indicated.



The discordant relationship between hsp28 protein and mRNA suggested that RA might regulate hsp28 protein posttranscriptionally. Analysis of the known amino acid sequence of hsp28 revealed several potential serine protease target sites. To determine whether hsp28 is a substrate of mbn, we isolated mbn from myeloblasts and purified it to homogeneity using a modification of the method described by Kao et al.(23) . (^2)The purity of the product was verified by mass spectrometry, N-terminal amino acid sequence determination, and SDS-PAGE (data not shown). mbn and rhsp28 were incubated at various substrate:enzyme ratios for 12 h at 37 °C, and then hsp28 protein degradation was determined by immunoblot analysis using an alpha-hsp28 mAb as the primary antibody. When incubated with mbn at an hsp28:mbn ratio of either 1:250 (Fig. 3, lane2) or 1:500 (lane3), the 28-kDa ``parent'' heat shock protein was almost completely hydrolyzed to two or three lower molecular weight immunoreactive bands (Fig. 3, arrowheads) corresponding to previously described hsp28 digestion products(9) . As a control, hsp28 did not undergo spontaneous degradation when incubated alone (Fig. 3, lane1). To further characterize the specificity of mbn proteolysis, we examined whether mbn also hydrolyzed proteins such as human Ig, bovine serum albumin, and lysozyme. Representative of these proteins was human IgG, which was not proteolytically cleaved by mbn (Fig. 4).


Figure 3: Proteolysis of hsp28 by mbn. Purified mbn and rhsp28 were incubated together for 12 h at 37 °C in a shaking water bath and then separated by 12% SDS-PAGE and transferred to a nitrocellulose filter. The filter was blotted with an alpha-hsp28 mAb (1:1500 dilution) and visualized using a goat alpha-mouse AP-conjugated secondary antibody (1:5000 dilution). Proteolysis was examined at an hsp28:mbn ratio (mol:mol) of 1:250 (lane2) and 1:500 (lane3). Arrowheads indicate the location of three new immunoreactive bands. As a control, rhsp28 (5 µg) was run in lane1. The relative position of molecular mass standards is indicated.




Figure 4: mbn does not hydrolyze human IgG. Purified human IgG was incubated either alone (lane1) or with mbn at a substrate:enzyme ratio of 1:500 (lane2) for 12 h at 37 °C in a shaking water bath and then separated by 12% SDS-PAGE and transferred to a nitrocellulose filter. Human IgG was immunoblotted using goat alpha-human IgG mAb (1:5000 dilution) and visualized using the ECL detection method. Human IgG was separated by SDS-PAGE under reducing conditions. The relative position of molecular mass standards is indicated.



Since mbn is homologous to human neutrophil elastase(6) , another protease down-regulated by RA during HL-60 cell differentiation, we examined the effect of hNE on hsp28 protein expression. rhsp28 and purified hNE were incubated together under conditions identical to that described above and were shown to hydrolyze elastin, a known substrate of hNE (data not shown). However, in contrast to mbn, rhsp28 was not affected by hNE (Fig. 5, lane2). rhsp28 alone was included as a control (lane3).


Figure 5: hsp28 is not degraded by human neutrophil elastase. rhsp28 was incubated with either myeloblastin (lane1) or hNE (lane2), in both cases at an hsp28:enzyme ratio of 1:500. Samples were separated by 12% SDS-PAGE and immunoblotted using an alpha-hsp28 mAb (1:1500 dilution) and visualized using a goat alpha-mouse horseradish peroxidase-conjugated secondary antibody (1:5000). Blots were developed using the ECL detection method. The arrowhead indicates the position of a complex of immunoreactive bands. rhsp28 (0.25 µg) was run as a control (lane3).



The mechanism by which decreased mbn protein signals cessation of growth and induction of cell differentiation remains unknown, in part due to the paucity of information regarding biological targets of this key protease. In this report, we identify hsp28 as the first reported protein substrate of mbn. As we have shown, this relationship is rather specific since hsp28 was not hydrolyzed by a homologous protease (Fig. 5), nor do these observations represent indiscriminate hydrolysis (Fig. 4). These findings are intriguing in light of the integral role played by hsp28 in physiological processes that directly impact on cell growth and differentiation. For example, cytoskeletal proteins are important components of signal transduction pathways. In fact, a number of growth regulatory molecules appear to exert their effects through interactions with cytoskeletal components(29, 30, 31) . In this context, hsp28 directly modifies the composition of actin microfilaments through its actin binding activity(22) . As a capping protein, the avian low molecular weight analogue of hsp28 inhibits actin polymerization(32, 33) . The effects of hsp28 on actin appear to be phosphorylation-dependent(22) , which is of interest in light of the identification of a serine kinase involved in the MAP kinase cascade being responsible for hsp28 phosphorylation(34, 35) . Originally associated with mitogen stimulation, activation of MAP kinases has also been linked to cell differentiation, including HL-60 cells(36, 37, 38, 39, 40) . Thus, RA appears to exert a dual effect on hsp28 protein expression through its down-regulation of mbn and activation of MAP kinases.

In addition, several members of the heat shock protein family, including hsp28, regulate peptide folding, intracellular protein trafficking, and signal transduction as molecular chaperones(21, 41, 42, 43, 44) . These interactions have been shown to modify the activity of associated proteins, which has significant consequences when the chaperoned protein is itself involved in growth regulation, as in the case of hsp90 and the oncogenic tyrosine kinase pp60(45, 46, 47) . Similarly, following transfection of baby rat kidney cells with either oncogenic or non-oncogenic adenovirus constructs, the presence of a 22-kDa protein associated with the rat low molecular weight hsp correlates inversely with oncogenicity of transformed cells(48) . Regulation of hsp28 by mbn could therefore directly impact on chaperonin activity.

Although the direct involvement of hsp28 in cell differentiation remains to be demonstrated, its identification as a target of mbn provides evidence that hsp28 is a component of the signal transduction pathway mediating differentiation of human myeloid leukemic cells. The identification of mbn in hematopoietic stem cells (49) suggests that a better understanding of mbn and its substrates may ultimately provide insight into the regulation of normal hematopoiesis.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA-40216-06 (to N. L. S.) and IF32CA08954-01 (to L. M. N.) and by a Terry Fox Development Grant from the National Cancer Institute of Canada (to W. H. M.). 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: Division of Hematology/Oncology, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, 1475 NW 12th Ave. (D8-4), Miami, FL 33136. Tel.: 305-548-4995; Fax: 305-548-5239.

(^1)
The abbreviations used are: RA, retinoic acid; hsp, heat shock protein; rhsp28, recombinant hsp28; mbn, myeloblastin; APL, acute promyelocytic leukemia; tRA, all-trans-retinoic acid; ECL, enhanced chemiluminescence detection system; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; MAP, mitogen-activated protein; TBS, Tris-buffered saline; hNE, human neutrophil elastase.

(^2)
J. L. Humes and E. Luedke, manuscript in preparation.


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