Transcriptional regulation of neurofilament expression by protein kinase A.
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 C alpha EV6 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 C alpha EV6 cells, activation of PKA by 80 microM ZnSO4 upregulated the expression of C alpha mRNA with maximal levels reached 8 hr post-treatment and maintained at 24 hr. Reporter gene assays in C alpha EV6 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.
White
L A
LA
Miami Project, University of Miami School of Medicine, Florida, USA.
Reeben
M
M
Saarma
M
M
Whittemore
S R
SR
D000818:Animals; D015152:Blotting, Northern; D002461:Cell Line, Transformed / Q000201:enzymology; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D007136:Immunoglobulins / Q000378:metabolism; D009414:Nerve Growth Factors / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016900:Neurofilament Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000201:enzymology; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D011903:Raphe Nuclei / Q000166:cytology; D051381:Rats; D014158:Transcription, Genetic / Q000502:physiology*
D017362:Cyclic AMP Response Element-Binding Protein; D007136:Immunoglobulins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; C090698:SP1 antigen; C044882:neurofilament protein L; C051774:neurofilament protein H; C061195:neurofilament protein M; D017868:Cyclic AMP-Dependent Protein Kinases