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Transcriptional regulation of neurofilament expression by protein kinase A
Author(s) -
White L.A.,
Reeben M.,
Saarma M.,
Whittemore S.R.
Publication year - 1997
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/(sici)1097-4547(19970201)47:3<242::aid-jnr2>3.0.co;2-i
Subject(s) - neurofilament , microbiology and biotechnology , cycloheximide , protein subunit , protein kinase a , cell culture , biology , downregulation and upregulation , transfection , transcriptional regulation , gene expression , protein biosynthesis , kinase , gene , biochemistry , genetics , immunohistochemistry , immunology
RN46A cells, a conditionally immortalized neuronal cell line derived from E12 rat medullary raphe nucleus, upregulate low M r (68 kDa, neurofilament [NF]‐L) and medium M r (160 kDa, NF‐M) neurofilament protein expression upon activation of protein kinase A (PKA). To examine possible transcriptional regulation of neurofilament protein expression by PKA, two cell lines were used; RN46A cells and CαEV6 cells, a cell line derived from RN46A cells that stably expresses the catalytic subunit of PKA under the control of the metallothionein promoter. Treatment of RN46A cells with dbcAMP resulted in an increase in the steady‐state levels of both NF‐L and NF‐M, but not high M r (200 kDa, NF‐H) neurofilament mRNA. These increases were both time and dose dependent and were sensitive to treatment with the protein synthesis inhibitor cycloheximide. In CαEV6 cells, activation of PKA by 80 μM ZnSO 4 upregulated the expression of Cα mRNA with maximal levels reached 8 hr post‐treatment and maintained at 24 hr. Reporter gene assays in CαEV6 cells following transfection with increasing lengths of the NF‐L promoter demonstrated that both a putative Sp1‐like and a cAMP response (CRE), but not a NGFI‐A, element were likely involved in PKA‐dependent activation of the NF‐L promoter. Electrophoretic mobility shift assays confirmed these results but showed that the nuclear proteins induced by PKA which bound to the NF‐L promoter Sp1‐like sequence were not Sp1. Collectively, these data suggest that constitutively expressed Sp1 may be involved in basal NF‐L promoter activity, and newly synthesized, PKA‐dependent nuclear proteins may synergistically activate the rat NF‐L promoter. J. Neurosci. Res. 47:242–252, 1997. © 1997 Wiley‐Liss, Inc.