May 18, 1993 - Expression of Recombinant HSP 27-related Danscripts in E. coli- ..... We also thank Dr. Bruce Baum and the National Institute of Dental.
THEJOIIRFAL OF B w u x : r x
Vol. 269. No. 11. Issue of March 1R. pp. 7869-7873, 1994 Printed 111 U.S.A.
CHEYISTK\
Differential Estrogenic Regulationof Small M , Heat Shock Protein Expression in Osteoblasts* (Received for publication, May 18, 1993, and in revised form, January 6 , 1994)
Lyndon F. Cooper$ and Katsumi Uoshima From the Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892
The rodent osteoblast cell culture model may provide a useful Polymerase chain reaction amplification and sequencing of heat shock protein (HSP) 27-related tran- s t u d y of HSP 27 expression caused by a variety of different scripts present in cDNAs generated from heat-shocked stimuli, including estrogen.The effect of cellular proliferation osteoblast RNA revealed the expression of three related o n HSP 27 expression was successfully evaluated in primary open reading frames that encode proteins of 208,197, or cultured rodent osteoblasts ( 5 ) . HSP 27 expression may be of 175 amino acids. Their expression as recombinant pro- particular significance to the estrogen-responsive osteoblast. teins in Escherichia coli demonstrated that they encode Cell culture, in addition to animal and human clinical studies, M , proteins. Northern blot anal- suggests that estrogen has marked effects on bone mass a n d 27,000, 25,000, or 22,000 ysis of heat shock protein 27-related transcript expres- osteoblast physiology (7). While estrogen responses have been sion revealed that, while HSP 27 expression (850-nucle- e v a l u a t e d i n s e v e r a l o s t e o b l a s t c u l t u r e m o d e l s ( 8 , 9), the obotide transcript) was induced by heat shock alone, the served effects of e s t r o g e n o n c e l l c u l t u r e m a y b e m o d e s t w h e n expression of a smaller transcript was facilitated by estrogen treatment priorto heat shock. This corresponded c o m p a r e d t o the clinical effectiveness of estrogen therapy on s effect of estrogen should be evaluM , proteins by b o n e m a s s ( 1 0 ) . P e r h a pthe with the co-inductionof 18,000 and 22,000 estrogen-pretreated, heat-shocked osteoblasts as re- ated in cells exposed to physical stress. Given the previous vealed by fluorography. Theidentification of these mul- observations of estrogenic induction of HSP 27 expression, the e effect of estrogen onHSP tiple small M , heat shock-induced proteins demon- a i m of this s t u d y w a s t o e x a m i n the strated that the stress response of mammalian cells is 27 expression by osteoblasts. composed of multiple, related proteins whose expresEXPERIMENTALPROCEDURES sion is differentially regulated. The importance ofestrogen-regulation of the small M , heat shock protein comCell Culture-The MC3T3-EI osteoblastic cell line (11)was cultured ponent of the stress response maybeof particular at 37 "C under a humidified atmosphere of 5% CO,, 95% air in RPMI 1640 medium containing 10% fetal calf serum and supplemented with significance to osteoblast physiology. insulin, transferrin, and selenium (Collaborative Research, and penicillidstreptomycin. HS was performed t o incubation of confluent cultures for 2 h at 43 "C in a humid, 5% CO,, 95% air atmosphere, Biological stress is presented to cells in various forms. Cel- followed by 4 h of recovery for protein analysis or 2 h of recovery for cells were grown in phelular responses to diverse stresses similarly involvethe expres- RNA analysis. For l7p-estradiol pretreatment, nol red-free media containing 0.34 fetal calf serum, insulin, transferrin, sion of several multigene families of HSP' that are grouped ac- selenium, and penicillidstreptomycin for 36 h, after which 17P-estracording totheir a p p a r e n t m o l e c u l a r w e i g h t t o i n c l uHSP d e 110, diol (100 p~ in ethanol) was addedto a final concentration of 20 nh! at HSP 90,HSP 70, and HSP 27 (1, 2). The v a r i o u s m e m b e r s of 0-24 h prior t o HS. PCR Amplification and Analysis of PCR Products-cDNA was first these multigene HSP families display distinct pattern of expressynthesized from 20 pg of total RNA using oligotdT1 (1.25 p i and sion. For example, HSP 70 isoforms are encoded by different ) Superscript (Life Technologies, Inc.) regenes expressed under non-stressed and stressed conditions (3). random primers (1.25 p ~ and verse transcriptase in 20-pl reactions incubated for 1 h at 42 "C. 15 p . ~ The m a m m a l i a n s m a l l M , HSP, HSP 27, i s e x p r e s s e d i n of each HSP 27-specific primer (upstream (LFCIS),5'-catgaccgagcggcT h i s p r o t e i nis also a r e s p o n s e t oHS in a variety of tissues (4). cgtcgtgc; downstream (LFCBO), 5"ttgcttctacttggctccaga) were used in constitutive product of select cell types ( 5 ) . F u r t h e r m o r e , 17p- standard 100-pl PCR reactions containing 2.5 units of Taq polymerase (AmplitaqT", Cetus)(12). cDNA was amplified using an initial cycle (5 estradiol treatment of breast tumor cells has been s h o w n t o elicit the expression of HSP 27 (6). Clearly, HSP 27 expression min, 95 "C; 5 min, 55 "C, 3 min, 72 "C) and 39 subsequent cycles (1min, 95 "C; 1 min, 55 "C, 1 min, 10 s; 72 T I . DNA products (20 pli were i s under complex physiologic regulation. The molecular basis separated by 1.0 or 1.6% agarose gel electrophoresis in Tris acetate for this is not defined. EDTA buffer and visualized by ultraviolet transillumination of ethidium bromide-stained DNA (13). Subcloning and Sequence Analysis-PCR products were eluted from * The costs of publication of this article were defrayed in partby the gels (Qiaex resin, Qiagen) andsubcloned into the EcoRV site of pBluepayment of page charges. This article must therefore be hereby marked script I1 KS' (pBSKS', Stratagene). Following transformation of these "advertisement" in accordance with 18 U.S.C. Section 1734 solely t o ligated products into Escherichia coli strain XL1-blue, alkaline lysis indicate this fact. The nucleotide seauence(s1 reuorted in this vaDer has been submitted miniprepplasmid DNA wasanalyzed by restrictiondigestionwith to the GenBankTh;/EMBLDataBankw>ti accession numberls) EcoRI and HindIII. Plasmids containing inserts of 500-700 bp were sequenced by a double-stranded, dideoxynucleotide strand termination UO3560-UO3562. method (14). Sequences were analyzed using the GCG Sequence Anali To whom correspondence should be addressed. Current address: ysis Software Package (15). School of Dentistry, CB 7450, Brauer Hall, University of North CaroExpression of Recombinant HSP 27-related Danscripts in E. colilina, Chapel Hill, North Carolina 27599-7450. The HSP 27 ORF were subcloned into BamHI and PstI sites of the Theabbreviationsusedare: HSP, heatshockprotein; HS, heat by PCR mutagenshock; ORF, open reading frame; IFTG, isopropyl-l-thio-p-o-galactopy- expression vector pQE9 (Qiageni. This was facilitated ranoside; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase esis of the 5' and 3' ends of the ORFto include a BamHI site andPstl a chain reaction; RT-PCR, reverse transcriptase-PCR; bp, base pair(s). site, respectively. The recombinant plasmidswere prepared as miniprep
7869
7870
Multiple Small
M,. Heat Shock Proteins
A 1
100
Hsp27a Hsp27b Hsp27c Hsp27ml
ATGACCGAGCGCCGCGTGCCCTTCTCGCTGCTGCGGACGCCGAGCTGGGgACCATTCCGGGACTGGTACCCTGCACACAGCCGCCTCTTCGATCAAGCTT ATGACCGAGCGCCGCGTGCCCTTCTCGCTGCTGCGGACGCCGAGCTGGGAACCATTCCGGGACTGGTACCCTGCACACAGCCGCCTCTTCGATCAAGCTT ATGACCGAGCGCCGCGTGCCCTTCTCGCTGCTGCGGACGCCGAGCTGGGAACCATTCCGGGACTGGTACCCTGCACACAGCCGCCTCTTCGATCAAGCTT ATGACCGAGC GCCGCGTGCCCTTCTCGCTGCTGCGGAGCCCGAGCTGGGAACCATTCCGGGACTGGTACCCTGCACACAGCCGCCTCTTCGATCAAGCTT LPC19>
Hsp27a H s p 2 7b Hsp27c H s p 2 7ml
TCGGGGTGCCCCGGTTGCCCGATGAGTGGTCGCAGTGGTTCAGCGCCGCTGGGTGGCCCGGATACGTGCGCCCGCTGCCCGCCGCGACCGCCGAGGGCCT TCGGGGTGCCCCGGTTGCCCGATGAGTGGTCGCAGTGGTTCAGCGCCGCTGGGTGGCCCGGATACGTGCGCCCGCTGCCC TCGGGGTGaC CC TCGGGGTGCCCCGGTTGCCCGATGAGTGGTCGCAGTGGTTCAGCGCCGCTGGGTGGCCCGGATACGTGCGCCCGCTGCCCGCCGCGACCGCCGAGGGCCC
Hsp27a Hsp27b Hsp27c Hsp27ml
C...GCGGTGACCCTaGCCGCACCAGCCTT GCCGCACCAGCCTT TGGCCGCACCAGCCTT CgcgGCGGTGACCCTGGCCGCACCAGCCTT
Hsp27a Hsp27b Hsp27c hsp27ml
301 GTGTCCCTGGACGTCAACCACTTCGCTCCGGAGGAGCTCACAGTGAAGAC GTGTCCCTGGACGTCAACCACTTCGCTCCGGAGGAGCTCACAGTGAAGAC GTGTCCCTGGACGTCAACCACTTCGCTCCGGAGGAGCTCACAGTGAAGAC GTGTCCCTGGACGTCAACCACGTGaTCCCG GAGGAGCTCACAGTGAAGAC
401 AACATGGCTACATCTCTCGGTGCTTCACCCGGAAATACACGCTCCCTCCAGGTGTGGACCCCACCCTAGTGTCCTCTTCCCTATCCCCTGAGGGCACACT AACA'TGGCTACATCTCTCGGTGCTTCACCCGGAAATACACGCTCCCTCCAGGTGTGGACCCCACCCTAGTGTCCTCTTCCCTATCCCCTGAGGGCACACT AACATGGCTACATCTCTCGGTGCTTCACCCGGAAATACACGCTCCCTCCAGGTGTGGACCCCACCCTAGTGTCCTCTTCCCTATCCCCTGAGGGCACACT AACATGGCTA CATCTCTCGGTGCTTCACCC GGAAATACAC GCTCCCTCCAGGTGTGGACCCCACCCTAGTGTCCTCTPCCCTATCCCCTGAGGGCACACT
500
Hsp27a Hsp27b Hsp27c Hsp27ml
501
600
Hsp27a Hsp27b Hsp27c Hsp27ml
TACCGTGGAG GCTCCGTTGC CCAAAGCAGT CACGCAGTCA GCGGAGATCA CCATTCCGGT TACTTTCGAG GCCCGCGCCC AAATTGGGGG CCCAGAAGCT TACCGTGGAG GCTCCGTTGC CCAAAGCAGTCACGCAGTCAGCGGAGATCACCATTCCGGTTACTTTCGAGGCCCGCGCCCAAATTGGGGGCCCAGAAGCT TACCGTGGAG GCTCCGTTGC CCAAAGCAGTCACGCAGTCAGCGGAGATCACCATTCCGGTTACTTTCGAGGCCCGCGCCCAAATTGGGGGCCCAGAAGCT TACCGTGGAG GCTCCGTTGC CCAAAGCAGT CACGCAGTCA aCGGAGATCA CCATTCCGGT TACTTTCGAG GCCCGCGCCC AAAWGGGGG CCCAGAAGCT
Hsp27a Hsp27b Hsp27c Hsp27ml
601 GGGAAGTCTG AACAGTCTGG AGCCAAGTAG AAGCCAT GGGAAGTCTG AACAGTCTGG AGCCAAGTAG AAGCCAT GGGAAGTCTG AACAGTCTGG AGCCAAGTAG AAGCCAT GGGAAGTCTG AACAGTCTGG AGCCAAGTAG AAGUT
101
200
.................... ........................................................................................ 300
201
................ ..............
CAGCCGAGCGCTCAACCGACAGCTCAGCAGCGGGGTCTCGGAGATCCGACAGACGGCTGATCGCTGGCGC CAGCCGAGCGCTCAACCGACAGCTCAGCAGCGGAGTCTCGGAGATCCGACAGACGGC'TGATCGCTGGCGC CAGCCGAGCGCTCAACCGACAGCTCAGCAGCGGGGTCTCGGAGATCCGACAGACGGCTGATCGCTGGCGC CAGCCGAGCGCTCAACCGAC AGCTCGGGGTCTCGGAGA TCCGACAGACGGCTGA
