Non-destructive analysis of museum objects by fibre-optic Raman spectroscopy Raman spectroscopy is a versatile technique that has frequently been applied for the investigation of art objects . By using mobile Raman instrumentation it is possible to investigate the artworks without the need for sampling . This work evaluates the use of a dedicated mobile spectrometer for the investigation of a range of museum objects in museums in Scotland , including antique Egyptian sarcophagi , a panel painting , painted surfaces on paper and textile , and the painted lid and soundboard of an early keyboard instrument . The investigations of these artefacts illustrate some analytical challenges that arise when analysing museum objects , including fluorescing varnish layers , ambient sunlight , large dimensions of artefacts and the need to handle fragile objects with care . Analysis of the musical instrument -LRB- the Mar virginals -RRB- was undertaken in the exhibition gallery , while on display , which meant that interaction with the public and health and safety issues had to be taken into account . Introduction Advances in analytical technology have led to increasing interest in the scientific examination of precious artefacts and antiquities . These examinations may be for a variety of reasons , most commonly fundamental interest in the materials and techniques used by the artist , or understanding their conservation , condition or degradation processes . Analytical data -LRB- e.g. from spectroscopic examinations -RRB- can , by material identification , assist in dating or authenticating art objects or works of art . The availability of these techniques is therefore very important and it enables us to solve specific questions concerning the conservation , restoration and history of artworks -LSB- 1 -- 3 -RSB- . In conservation science , analytical techniques which are non-destructive or micro-destructive are particularly important . Raman spectroscopy is such a technique , allowing material identification from particles down to 1 μm . This method has already been used for the investigation of different artefacts , including panel paintings -LSB- 4 , 5 -RSB- , glass -LSB- 6 -RSB- , wall paintings -LSB- 7 , 8 -RSB- , manuscripts -LSB- 9 -- 11 -RSB- and historical biomaterials -LSB- 12 , 13 -RSB- . Although this technique used to be expensive and highly specialised , it is becoming increasingly more accessible to large museums owing to instrumental improvements and lower costs -LSB- 14 -- 17 -RSB- . However , regional museums often still do not have access to this kind of analytical facility . Use of mobile equipment is one solution to this problem , since several institutions may benefit from a single instrument . In addition , mobile fibre-optic-based Raman instrumentation may facilitate the analysis of artefacts that are difficult or impossible to bring to the laboratory , such as wall paintings on the vault of a chapel -LSB- 8 -RSB- . In this work we evaluate the use of a mobile fibre-optic-based Raman instrument to perform investigations in a museum context . Different types of artefacts were investigated in a variety of locations , testing and demonstrating the flexibility in instrumental set-up . In particular the experimental conditions during the examination of a virginals on exhibition were challenging . Instrument stability after travelling overseas -LRB- between Belgium and Edinburgh , UK -RRB- was also thoroughly evaluated . Experimental Objects were investigated in the conservation and research laboratories , stores and an exhibition gallery of the National Museums of Scotland -LRB- NMS -RRB- , Edinburgh , as well as in the Burrell Collection -LRB- Glasgow -RRB- -LRB- 31 October -- 13 November 2005 -RRB- . For these surveys mobile Raman equipment was used , which has been described extensively elsewhere -LSB- 19 -RSB- . The core of the equipment consists of a SpectraPro-150i 150-mm spectrometer and a thermoelectrically cooled charge-coupled device detector -LRB- Roper Scientific/Princeton Instruments -RRB- . The system is also equipped with a 785-nm diode laser for excitation , which has a maximal output power of 300 mW at the source . In order to avoid damaging the artefacts during these investigations , and depending on the object and the colour of the area under investigation , the output power was limited to a maximum of 10 mW -LRB- measured at the surface -RRB- . The accumulation time ranged from 10 s to 1 h for weak Raman scatterers . For these investigations the 600 lines/mm grating was selected . Due to non-ideal focussing conditions , the expected minimal laser spot size of ca. 25-μm diameter -LRB- when working with a × 6 infinity-corrected objective lens -RRB- could not be reached . In practice we worked with a larger spot size of ca. 50 μm . For each area at least 10 spectra were recorded and evaluated in order to tackle paint inhomogeneity at this scale . The experimental configuration was adapted to suit the artefact under study . For example , the probe head could either be mounted horizontally or vertically on the articulating arm and extension tubes could be used on the probe head to facilitate access to the artworks -LSB- 8 , 18 -RSB- . Results and discussion When testing the versatility of a mobile spectrometer in a museum context , the technique needs to be applied to a broad range of artefacts and questions . Throughout this text we will provide several examples , illustrating different facets of this approach . Table 1 gives an overview of the investigated artefacts , together with the analytical challenges that these investigations represent . Table 1Overview of the different artefacts that were investigated in this work and the analytical challenges they representInvestigated artefactAnalytical challengeEgyptian sarcophagus -LRB- NMS -RRB- -LRB- A. 1907.569 A -RRB- Pigment identification through a thick varnish layerEgyptian sarcophagus -LRB- Glasgow -RRB- -LRB- 1895.167 . a -RRB- Pigment analysis with strong interference from sunlight . Transport to a different museum , analyse , and return in 1 dayDean panel -LRB- H.KL 72 -RRB- Analysis of a varnished paintingKalighat painting -LRB- A. 1912.122 -RRB- Analysis of work on paperIndian textile banner -LRB- K. 2005.234 -RRB- Analysis of a textile artefact of large dimensions with a thin and friable painted decorationMar virginals -LRB- H.LT 122 -RRB- Investigation in the exhibition gallery open to the public The examination of Egyptian sarcophagi , both in the National Museums of Scotland -LRB- Edinburgh -RRB- and in the Burrell Collection -LRB- Glasgow Museums & Art Gallery -RRB- illustrate the comparative ease of transportation of the instrument . The capability of the technique to analyse pigments on artefacts covered by a thick varnish layer as well as to undertake the investigation with interference from sunlight are shown . During the examination of a Renaissance painted panel -LRB- one of the panels from Dean house , Edinburgh -RRB- , strong inteference from a fluorescing varnish layer was also encountered . The possibilities of the technique for the non-invasive investigation of fragile artefacts on paper are illustrated by the yellow pigment identification on Indian Kalighat paintings and on a painted textile banner . During the latter survey the articulating arm was used to allow analysis of a large artefact displayed horizontally . Finally , working in the galleries represented an interesting experience as it involved undertaking analytical research whilst taking into account ambient conditions and visitors ' curiosity . This approach is illustrated by the study of the Mar virginals . Pigment analysis of antique Egyptian sarcophagi An antique Egyptian sarcophagus -LSB- A. 1907.569 A -RSB- , dating from the twenty first/second Dynasty -LRB- 1069 -- 715B.C . -RRB- , was investigated at the NMS by using mobile Raman spectroscopy -LRB- Fig. 1a -RRB- . Samples from this artefact had been examined before by Fourier transform -LRB- FT -RRB- Raman spectroscopy -LSB- 19 -RSB- . The aim of the present study was firstly to investigate whether non-destructive mobile Raman spectroscopy confirmed the previous findings . Secondly we wished to investigate the thick yellowed varnish layer , to see how it influenced the investigation , to establish when it was most likely to have been applied and what its visual effect was on the underlying colours . Fig. 1a Experimental set-up for the investigation of an Egyptian sarcophagus at the NMS . b Stack plot of the baseline-corrected Raman spectrum obtained from the yellow areas of this sarcophagus and the reference spectrum of orpiment -LRB- As2S3 -RRB- . -LRB- Experimental conditions for the spectrum of the artefact : 60 accumulations of 1 s , × 6 objective , 785 nm , ca. 5 mW at the surface -RRB- As expected , the yellow varnish generated serious interference of fluorescence radiation which overwhelmed the weaker Raman signal . Unfortunately it was not possible to identify its composition and therefore determine if it was original or whether it was the result of a previous conservation treatment . Nevertheless different pigments on the artefact could be identified . Figure 1b shows that the baseline-corrected Raman spectrum of the omnipresent yellow pigment on this sarcophagus is orpiment -LRB- As2S3 -RRB- . Other pigments that could be identified were hematite -LRB- Fe2O3 -RRB- , calcite -LRB- CaCO3 -RRB- and carbon black -LRB- C -RRB- . This is in agreement with the findings by Eremin et al. -LSB- 19 -RSB- . Although positive results were obtained , it was impossible to record a satisfactory Raman spectrum of the green and blue painted areas of the sarcophagus . It is very likely that Egyptian blue was used in this artefact ; however , when stimulated with a 785-nm laser this pigment does not yield a Raman spectrum but gives rise to serious fluorescence background . Eremin et al. -LSB- 19 -RSB- indeed identified Egyptian blue with FT Raman spectroscopy -LRB- although the sample did not come from exactly the same area -RRB- , so this illustrates one of the disadvantages of using mobile equipment . In contrast to many laboratory instruments , mobile instrumentation is usually equipped with a single laser , which makes it impossible to switch to another laser wavelength to overcome fluorescence interference . A second Egyptian sarcophagus -LSB- 1895.167 . a -RSB- was investigated at the Burrell Collection in Glasgow , illustrating one of the advantages of using mobile equipment : several collections can share one instrument . The sarcophagus -LRB- Fig. 2a -RRB- , dating from the ancient Egyptian New Kingdom -LRB- 1570 -- 1070B.C . -RRB- , was in the conservation lab , with high levels of natural light . The conservator wished to know the identity of the yellow pigment on the cover of the sarcophagus , since orpiment -LRB- As2S3 -RRB- may degrade when exposed to high levels of light . Figure 2b shows that the yellow pigment on the sculptured head and hands of the sarcophagus lid indeed was indeed orpiment -LRB- As2S3 -RRB- , whereas the yellow pigment on the other areas was limonite -LRB- FeOOH · nH2O -RRB- . Fig. 2a Experimental set-up for the investigation of an Egyptian sarcophagus in the conservation lab of the Burrell Collection , Glasgow . Ambient sunlight interfered with the investigations . b Stack plot of two Raman spectra obtained from the sculptured head and hands of this sarcophagus . -LRB- Experimental conditions : × 6 objective , 785 nm , ca. 5 mW at the surface , 120 accumulations of 1 s -RRB- Pigment investigation of a painted panel from Dean House , Edinburgh Mobile Raman spectroscopy was applied to identify the pigments in one of the Renaissance painted panels from Dean House in Edinburgh . The `` Deans Panels '' are thought to date from around 1627 -LSB- 20 -RSB- . The panel representing the sense of hearing -LRB- KL 72 -RRB- is exhibited on a wall , at a height of about 3 m , which would have required the construction of scaffolding in the gallery . Therefore it was considered more practical to remove the panel temporarily from the exhibition and bring it to the conservation lab for analysis . The painted surface was found to be covered with a varnish layer , which contributed to the fluorescence background , although it still was possible to identify the pigments in the investigated areas of the artefact . Vermilion -LRB- HgS -RRB- was observed in the red areas , whereas orange areas merely contained red lead -LRB- Pb3O4 -RRB- . Brownish red areas were painted with a hematite -LRB- Fe2O3 -RRB- - containing paint . On several occasions , mixtures of two of these pigments were encountered . Black areas contained carbon black -LRB- C -RRB- , whereas the white regions were painted by using lead white -LRB- 2PbCO3 · Pb -LRB- OH -RRB- 2 -RRB- . Pink zones consisted of a mixture of the latter pigment with vermilion ; vermilion could also be identified in the purple regions -LRB- the blue component of which could not be identified -RRB- . Finally , the yellow areas examined were painted by using lead -- tin yellow , type I -LRB- Pb2SnO4 -RRB- . All these pigments are consistent with the early modern origin of the artwork . Identification of the yellow pigment from an Indian Kalighat painting The yellow pigment from different areas from an Indian Kalighat painting was examined by using mobile Raman spectroscopy . These are nineteenth century Indian watercolour and opaque media paintings on industrial paper . Since paper artefacts are fragile , special care has to be taken not to damage them during handling and positioning of the spectrometer . Gloves were worn and positioning of the probe head was performed with great care . On all the yellow painted areas of the Kalighat paintings that were examined , the same yellow pigment was encountered . Figure 3 shows the Raman spectrum obtained , along with the reference spectrum of chrome yellow -LRB- PbCrO4 -RRB- . It is clear that the yellow pigment in the Kalighat paintings is chrome yellow . Fig. 3Stack plot of the Raman spectra obtained from a yellow area from an Indian Kalighat painting and the reference spectrum of chrome yellow -LRB- PbCrO4 -RRB- . -LRB- Experimental conditions for the spectrum of the artefact : 8 accumulations of 150 s , × 6 objective , 785 nm , ca. 3 mW at the surface -RRB- Analysis of a painted banner The National Museums of Scotland has a large -LRB- 1,080 × 2,030 mm -RRB- textile banner of uncertain provenance in its collection . Stylistic interpretations of the polychrome design suggest an Indian origin . The banner depicts , on a green background , colourful imagery of the different stages on the way to heaven -LRB- Fig. 4b , detail -RRB- . Because of its large dimensions and the absence of appropriate non-destructive analytical methods , no previous investigation had been made and little was known of the materials that have been applied . The mobile Raman spectrometer was therefore used to identify the pigments . Fig. 4a Experimental set-up for the investigation of a large textile banner . b Lower part of the investigated textile banner . c Raman spectrum of an orange area of the textile banner , together with the reference spectra of massicot -LRB- PbO -RRB- and red lead -LRB- Pb3O4 -RRB- . -LRB- Experimental conditions for the spectrum of the artefact : 1 accumulation of 150 s , × 6 objective , 785 nm , ca. 10 mW at the surface -RRB- . d Raman spectrum of a red area of the textile banner and reference spectrum of vermilion . -LRB- Experimental conditions : 1 accumulation of 150 s , × 6 objective , 785 nm , ca. 5 mW at the surface -RRB- In order to be able to examine a range of positions on the surface of the banner , the articulating arm was used for macro-positioning of the probe head , while the artefact was placed horizontally on a table -LRB- Fig. 4a -RRB- . All the paint surfaces were thin , and in several areas the paint was friable or had already been lost . Red areas gave rise to the Raman spectrum of vermilion -LRB- HgS , Fig. 4d -RRB- , whereas the orange areas yielded a more complex spectrum , which consists of a combination of massicot -LRB- PbO -RRB- and red lead -LRB- Pb3O4 -RRB- -LRB- Fig. 4c -RRB- . Red lead is an orange pigment , whereas massicot has a more yellowish shade . It is not clear whether massicot was intentionally added to the paint or whether the latter pigment arose as a side-product of the synthesis of red lead . Other pigments that were positively identified in the banner were calcite -LRB- CaCO3 -RRB- , carbon black -LRB- C -RRB- , lead white -LRB- 2PbCO3 · 2 H2O -RRB- , azurite -LRB- 2CuCO3 · Cu -LRB- OH -RRB- 2 -RRB- and anatase -LRB- TiO2 -RRB- . Under the ambient conditions of the conservation laboratory , which did not have full light exclusion , and using the 785-nm laser , it was impossible to obtain a spectrum of sufficient quality to identify the green areas on the artefact . One solution to overcome this problem would be to use a Raman instrument with a different laser , with a smaller absorption cross section for the green pigments . Investigation of an early keyboard instrument , the Mar virginals The Mar virginals is an early keyboard instrument , which , although probably made in the Low Countries , has a long association with Lady Mary Stewart , countess of Mar and could have been made in Scotland . It probably dates from between A.D. 1560 and 1660 , and its lid and soundboard contain three separate painted panels , the central one depicting Orpheus playing to the animals . We wished to compare the paintings on the lid to those on the soundboard , since similar or different pigment compositions may support or disprove the idea that the lid is contemporary to the virginals . This artefact had to be analysed while on display in the museum gallery , which enabled us to evaluate the feasibility of undertaking the analytical work in a public space . It was necessary to ensure that there were no health and safety risks , especially from direct or scattered light from the laser beam , as well as keeping the electronics and power supply cables and units away from enquiring fingers . The visiting public were clearly intrigued with what was going on and to help explain this , a poster informed the visitors about the experiments . Working in the gallery also meant that the ambient conditions were far from ideal . During this study , two issues severely hampered the Raman investigations : background light and spatial limitations . It was not possible to reduce the intensity of background light to avoid interference which tended to overwhelm the spectrum , nor on this occasion to undertake the work overnight . And since the Mar virginals was not taken out of its display case , little space was available to align the probe head with the painted surface , the cramped conditions meaning that great care had to be taken not to touch the artefact . To maximise flexibility the probe head was mounted on an articulating arm and extension tubes -LSB- 8 -RSB- were used to approach the painted surface -LRB- Fig. 5 -RRB- . Fig. 5Experimental set-up for the investigation of the Mar virginals Nevertheless , despite these non-ideal conditions , the pigments could be identified . White lead -LRB- 2PbCO3 · Pb -LRB- OH -RRB- 2 -RRB- is omnipresent : almost all the recorded Raman spectra contain the intense Raman band at 1,056 cm − 1 , whereas the less intense bands of white lead at 260 , 203 and 154 cm − 1 are only observed in a few spectra . Apparently , this pigment was applied to modify the hue of the coloured areas , making them appear brighter . From the intense Raman bands at 254 , 283 and 343 cm − 1 , it is clear that the red areas were painted with the red pigment vermilion -LRB- HgS -RRB- , whereas the bright yellow areas were painted by using orpiment -LRB- As2S3 -RRB- . Carbon black -LRB- C -RRB- is the black pigment which was used in the painted decoration . In general , the blue areas of the painting did not yield Raman spectra of sufficient quality to allow identification . The reasons for this are multiple . In general , blue and green pigments absorb the red -LRB- 785 nm -RRB- laser light which hinders the recording of a spectrum . In addition there was a large amount of background radiation , both from the gallery lights and from fluorescence , which was probably caused by the varnish layer . It is very likely that the blue pigment azurite -LRB- 2CuCO3 · Cu -LRB- OH -RRB- 2 -RRB- is used in the artwork , since this pigment was very commonly used in Renaissance period : however , unfortunately azurite happens to be a weak Raman scatterer when using a 785-nm laser , which means that small amounts of background radiation easily overwhelm the Raman spectrum . Although only a few areas on the soundboard could be examined , the pigments on the lid were in good agreement with those on the soundboard . Despite the fact that this research project should be considered as a pilot study , this tends to support the thesis that the lid and the soundboard originate from the same instrument , or at least were painted at a similar period . However other approaches , such as stylistic studies , or taking samples to matching the spore-element compositions of the paint from the lid and the soundboard -LSB- 9 -RSB- , are needed to be more definite . Since the investigation of this rare musical instrument was performed in one day , time was confined and only a limited number of areas could be examined . In fact , except for the absence of lead -- tin yellow , none of the pigments were rare for an artwork of this period . Conclusions This work reports on the application of mobile Raman spectroscopy to the non-invasive investigation of a range of object of arts in a museum context . The different cases presented here illustrate different obstacles and analytical challenges that are often encountered during direct Raman investigations of museum objects . The investigation of two antique Egyptian sarcophagi shows the possibilities of the technique to deal with artefacts covered by a thick varnish layer as well as the investigation with the interference of ambient sunlight in the conservation laboratory . The investigation of the Dean House panel illustrates the possibilities of pigment investigation although a fluorescing varnish layer is present ; the suitability of the method to analyse fragile artefacts was demonstrated by the investigation of Indian Kalighat paintings on paper ; the textile banner was both fragile and large . Finally , the Mar virginals has been examined for the first time directly in the exhibition gallery with normal lighting conditions and with visitors present . All these examples illustrate that non-destructive , mobile Raman spectroscopy is able to cover a broad field of research questions on artistic and historical objects , in a range of different experimental conditions , encountered in a museum context .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

raman spectroscopy
conservation science
mobile raman equipment

art analysis
non-destructive investigation


Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes : Effects of endothelin-1 , oxidative stress and cytokines Krüppel-like transcription factors -LRB- Klfs -RRB- modulate fundamental cell processes . Cardiac myocytes are terminally-differentiated , but hypertrophy in response to stimuli such as endothelin-1 . H2O2 or cytokines promote myocyte apoptosis . Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes . We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes . In response to endothelin-1 , Klf2 mRNA expression was rapidly increased -LRB- ∼ 9-fold ; 15 -- 30 min -RRB- with later increases in expression of Klf4 and Klf6 -LRB- ∼ 5-fold ; 30 -- 60 min -RRB- . All were regulated as immediate early genes -LRB- cycloheximide did not inhibit the increases in expression -RRB- . Klf5 expression was increased at 1 -- 2 h -LRB- ∼ 13-fold -RRB- as a second phase response -LRB- cycloheximide inhibited the increase -RRB- . These increases were transient and attenuated by U0126 . H2O2 increased expression of Klf2 , Klf4 and Klf6 , but interleukin-1β or tumor necrosis factor α downregulated Klf2 expression with no effect on Klf4 or Klf6 . Of the Klfs which repress transcription , endothelin-1 rapidly downregulated expression of Klf3 , Klf11 and Klf15 . The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that , as a family , they are actively involved in regulating phenotypic responses -LRB- hypertrophy and apoptosis -RRB- to extracellular stimuli . 1 Introduction C2H2 zinc finger transcription factors possess two cysteine and two histidine residues which co-ordinate Zn2 + within each finger to form a conserved DNA-binding structure . Sp1-like proteins and Krüppel-like factors -LRB- Klfs -RRB- each have three C2H2 zinc fingers at their C-termini , and Klfs are distinguished by a highly conserved interfinger space sequence -LSB- 1 -RSB- . The seventeen mammalian Klfs which have been identified are now designated Klf1-17 -LSB- 2,3 -RSB- , though many have alternative names . Several were named according to the tissues in which they were originally shown to be enriched -LRB- e.g. Klf1 is erythroid Eklf , Klf2 is lung Lklf , Klf4 is gut Gklf , Klf5 is intestinal Iklf and Klf15 is kidney Kklf -LSB- 2 -RSB- -RRB- . Other Klfs are widely expressed including Klf3 -LRB- basic Bklf -RRB- , Klf6 and Klf7 -LRB- ubiquitous Uklf -RRB- . Klf9 was identified as a basal transcription element binding -LRB- BTEB -RRB- protein , with Klf5 and Klf13 as homologues -LRB- BTEB2 and BTEB3 , respectively -RRB- . Klf10 and Klf11 were identified as early genes induced by transforming growth factor β and were named TIEG and TIEG2 , respectively . Klfs bind to consensus GC-rich or CACCC sequences through the three C-terminal zinc fingers -LSB- 1 -RSB- . The frequency of such sequences in gene promoters , coupled with the high sequence identity between Klf proteins in their DNA-binding domains , raises questions relating to the specificity of any Klf for a particular promoter and potential functional redundancy . Nevertheless , Klfs can be clustered according to whether they act primarily as transcriptional repressors and/or activators , and the domain structure of the N-terminal regulatory regions . Klf3 , Klf8 and Klf12 recruit C-terminal binding proteins -LRB- CtBPs -RRB- to repress transcription , whereas Klf9 , Klf10 , Klf11 , Klf13 and Klf16 repress transcription through interaction with mSIN3a -LSB- 1,4 -RSB- . Klf15 also represses transcription , though the mechanism is not clear . Klf2 , Klf4 , Klf5 , Klf6 , and Klf7 are largely transcriptional activators -LSB- 1 -RSB- , but may suppress gene expression in specific circumstances -LRB- e.g. Klf5 negatively regulates expression of Klf4 -LSB- 5 -RSB- -RRB- . In addition to interactions with other proteins , post-translational modifications -LRB- e.g. phosphorylation or acetylation -RRB- regulate the transactivating activities of different Klfs -LSB- 1,6 -RSB- . Globally , Klfs regulate fundamental cellular responses such as growth , apoptosis , angiogenesis and proliferation . For example , Klf5 overexpression is associated with cell proliferation , whereas Klf2 , Klf4 and Klf6 are more consistently implicated in cell cycle arrest -LSB- 7 -- 9 -RSB- . Klf4 and Klf5 may both promote apoptosis induced by oxidative stress though the mechanism is unclear -LSB- 9 -RSB- . Mammalian cardiac myocytes -LRB- the contractile cells of the heart -RRB- become terminally-differentiated shortly after birth . Subsequent growth of the heart results from an increase in size of individual cardiac myocytes and , in the adult , myocytes may hypertrophy in order to maintain or increase cardiac output . This is associated with physiological and morphological changes -LRB- increases in cell size and myofibrillogenesis -RRB- and changes in gene expression including increased expression of immediate early genes -LRB- IEGs -RRB- , and re-expression of genes normally expressed in early development -LSB- 10 -RSB- . Stimuli such as endothelin-1 -LRB- ET-1 -RRB- , which activate Gq protein-coupled receptors are particularly implicated in cardiac myocyte hypertrophy , and promote both the physiological/morphological changes and the changes in gene expression associated with the response -LSB- 11 -RSB- . In contrast , oxidative stresses or pro-inflammatory cytokines may induce cardiac myocyte apoptosis -LSB- 12 -RSB- . Many studies have examined the intracellular signalling pathways which are activated by various stimuli in cardiac myocytes , and these are presumed to lead to changes in gene and protein expression to promote hypertrophy or to facilitate myocyte death -LSB- 13 -RSB- . Many Klfs are expressed in adult hearts to a degree although the cell types in which they are expressed can not be ascertained . For example , Klf2 is associated with endothelial cells -LSB- 14,15 -RSB- , and cardiac endothelial cells could account for expression of Klf2 in whole heart extracts -LSB- 16 -RSB- . As reviewed by Haldar et al. -LSB- 17 -RSB- , Klf13 and Klf15 are the only Klfs which have so far been shown to play a role in cardiac myocytes . Klf13 is highly expressed in adult hearts -LSB- 18 -RSB- and is required for normal cardiac development in Xenopus -LSB- 19 -RSB- . Klf15 is highly expressed in adult hearts and in cardiac myocytes -LSB- 20,21 -RSB- . It appears to be anti-hypertrophic since it is downregulated during hypertrophy , and overexpression of Klf15 in cardiac myocytes suppresses the morphological changes and changes in gene expression induced by hypertrophic stimuli -LSB- 21 -RSB- . Our microarray studies highlight the dynamic changes in expression of many genes -LRB- including Klfs -RRB- induced by H2O2 -LRB- an example of oxidative stress -RRB- or ET-1 in cardiac myocytes over 4 h -LSB- 22 -- 25 -RSB- . Here , we explore further the regulation of Klf family members in cardiac myocytes in response to ET-1 , H2O2 and pro-inflammatory cytokines -LSB- interleukin 1β -LRB- IL-1β -RRB- and tumour necrosis factor α -LRB- TNFα -RRB- -RSB- . The dynamic regulation of expression of multiple Klf family members suggests that , as a family , they are actively involved in regulating phenotypic responses of cells to extracellular stimuli . 2 Materials and methods 2.1 Primary culture of neonatal rat cardiac myocytes and preparation of polysomes Myocytes were dissociated from the ventricles of 2 - to 4-day-old Sprague‑Dawley rat hearts by an adaptation of the method of Iwaki et al. -LSB- 26 -RSB- as previously described -LSB- 27 -RSB- . Cells were plated at 4 × 106 cells/60 mm Primaria culture dish -LRB- BD Biosciences -RRB- for 18 h in 15 % -LRB- v/v -RRB- foetal calf serum , then serum was withdrawn for 24 h before experimentation . Myocytes were exposed to ET-1 -LRB- 100 nM -RRB- , H2O2 -LRB- 0.2 mM -RRB- , IL-1β -LRB- 100 ng/ml -RRB- or TNFα -LRB- 10 ng/ml -RRB- with or without pre-treatment -LRB- 10 min -RRB- with inhibitors -LRB- 10 μM U0126 ; 50 μM LY294002 ; 5 μM SB203580 -RRB- . Cardiac myocyte polysomes were prepared by sucrose density centrifugation -LRB- 0.8 -- 1.6 M sucrose gradients ; 5 ml -RRB- as previously described -LSB- 25 -RSB- . Fractions were collected by upward displacement -LRB- fraction 1 from the top of the gradient , 0.8 M sucrose ; fraction 12 from the bottom of the gradient , 1.6 M sucrose -RRB- whilst monitoring absorbance at 254 nm . 2.2 RNA preparation and quantitative PCR -LRB- qPCR -RRB- Total RNA and polysome RNA -LRB- fractions 6 -- 11 of the sucrose gradients -RRB- were extracted and cDNA synthesized using reverse transcription as previously described -LSB- 22,25 -RSB- . qPCR was performed using a Real‑Time PCR System -LRB- Model 7500 , Applied Biosystems -RRB- as described -LSB- 25 -RSB- using primers designed to amplify mRNA sequences across an intron -LRB- Table 1 -RRB- . qPCR analysis of glyceraldehyde 3-phosphate dehydrogenase -LRB- Gapdh -RRB- was performed as a control and the relative quantification protocol was used . PCR conditions were 50 °C for 2 min , 95 °C for 10 min -LRB- Jump-Start Taq polymerase activation step -RRB- , followed by 40 cycles of 95 °C for 15 s and 59 °C for 60 s. Following qPCR , dissociation curve analysis was routinely performed to check for aberrant amplification products -LRB- e.g. primer‑dimers -RRB- . 2.3 Western blotting Cardiac myocyte nuclear extracts were prepared and Western blotting performed essentially as described -LSB- 28 -RSB- . Extracts from 1.8 × 106 cells were analysed with separation of proteins on 10 % -LRB- v/v -RRB- polyacrylamide gels . Nitrocellulose blots were probed with Klf6 rabbit polyclonal antibodies -LRB- Santa Cruz Biotechnology Inc. ; Klf6 -LRB- R-173 -RRB- , sc-7158 , 1/500 dilution -RRB- . Bands were detected and analysed by scanning densitometry as described -LSB- 29 -RSB- . 3 Results 3.1 ET-1 regulates expression of multiple klf family members in cardiac myocytes Our microarray studies -LRB- with Affymetrix rat genome 230 2.0 arrays -RRB- of the effects of ET-1 on cardiac myocyte gene expression over 4 h -LSB- 25 -RSB- identified Klfs as a family of transcription factors which appeared to be dynamically regulated at the mRNA level . Mining the data specifically for Klfs , we identified rapid and transient increases in expression of Klf2 , Klf4 , Klf5 , Klf6 , Klf9 and Klf10 , with concomitant downregulation of Klf3 , Klf11 and Klf15 -LRB- Table 2 -RRB- . There was no statistically significant change in expression of Klf13 or Klf16 , although Klf13 exhibited a small -LRB- non-significant ; one-way ANOVA with Tukey post-test -RRB- increase in expression over 2 -- 4 h. Klf1 -LRB- probeset 1382033_at -RRB- , Klf7 -LRB- probesets 1380363_at , 1377618_at , 1384497_at -RRB- and Klf12 -LRB- probeset 1385545_at -RRB- were not consistently called `` present '' with low fluorescence values and are thus expressed at minimal levels . We could not identify Klf8 , Klf14 -LRB- Sp6 -RRB- or Klf17 on the arrays . Using qPCR , we examined more carefully the effects of 100 nM ET-1 on Klf mRNA expression . Klf2 mRNA expression was significantly increased within 15 min , with maximal expression -LRB- ∼ 9-fold relative to controls -RRB- at 30 min -LRB- Fig. 1A -RRB- . Klf4 and Klf6 mRNAs were also upregulated -LRB- Fig. 1 , B and C -RRB- , though the relative stimulation was less -LRB- ∼ 5-fold relative to controls -RRB- and the response was delayed relative to Klf2 -LRB- maximal expression at 0.5 -- 1 h -RRB- . Klf5 mRNA was upregulated from 1 h , with maximal expression at 1.5 h -LRB- ∼ 13-fold relative to controls -RRB- -LRB- Fig. 1D -RRB- . For these Klfs , the increase in expression was transient , with expression of Klf2 returning to baseline within 90 min and expression of Klf4 , Klf5 and Klf6 declining to basal levels within 4 h . The increases in expression of Klf9 and Klf10 were relatively small -LRB- maximal expression < 2-fold at 1.5 -- 2 h -RRB- although levels remained elevated over 4 -- 6 h -LRB- Fig. 1 , E and F -RRB- . Consistent with our microarray data , Klf3 , Klf11 and Klf15 were downregulated in response to ET-1 . Whereas the decrease in expression of Klf3 was transient with minimal expression at ∼ 30 min -LRB- Fig. 1G -RRB- , the decrease in expression of Klf11 and Klf15 was sustained over at least 6 h -LRB- Fig. 1 , H and I -RRB- . It is notable that the Klfs which were downregulated and those with only a small increase in expression are largely associated with transcriptional repression , whereas the Klfs with the greatest increases in expression -LRB- Klf2 , Klf4 , Klf5 and Klf6 -RRB- are mostly associated with transcriptional activation -LSB- 1,4,6 -RSB- . Further studies focused on this latter group . 3.2 Regulation of Klf2 , Klf4 , Klf5 and Klf6 by ET-1 To determine whether Klf2 , Klf4 , Klf5 and Klf6 mRNAs were upregulated as IEGs -LRB- i.e. the increase in expression is regulated by pre-existing transcription factors and does not require synthesis of new proteins -RRB- , we examined the effects of 20 μM cycloheximide -LRB- to inhibit protein synthesis -RRB- . Cycloheximide alone increased the expression of Klf2 , Klf4 and Klf6 mRNAs -LRB- Fig. 2A ; probably due to activation of stress-activated signalling pathways -LSB- 30,31 -RSB- -RRB- , but did not inhibit the increase in expression induced by ET-1 . However , the increase in expression of Klf5 by ET-1 was inhibited by cycloheximide -LRB- Fig. 2B -RRB- , indicating that de novo protein synthesis is required for upregulation of Klf5 mRNA . Thus , Klf2 , Klf4 and Klf6 are regulated as IEGs in response to ET-1 , whereas Klf5 is a second phase gene . ET-1 potently activates the extracellular signal-regulated kinases 1/2 -LRB- ERK1/2 -RRB- in cardiac myocytes -LSB- 32 -RSB- . We used 10 μM U0126 -LRB- a selective inhibitor of the ERK1/2/5 cascades -LSB- 33 -- 35 -RSB- , though ERK5 is not significantly activated by ET-1 -LSB- 24 -RSB- -RRB- to determine whether the increase in expression of Klf2 , Klf4 , Klf5 and Klf6 requires ERK1/2 signalling . U0126 minimally affected basal expression of each Klf , but partially inhibited the ET-1-induced increase in expression of Klf2 -LRB- 56 ± 4 % inhibition at 30 min ; mean ± SEM , n = 5 -RRB- , Klf4 -LRB- 43 ± 9 % inhibition at 1 h ; n = 4 -RRB- , Klf5 -LRB- 72 ± 8 % inhibition at 1 h ; n = 4 -RRB- and Klf6 -LRB- 45 ± 8 % inhibition at 1 h ; n = 4 -RRB- -LRB- Fig. 3A -RRB- . These data suggest that ERK1/2 signalling is required , at least in part , for the upregulation of Klf2 , Klf4 , Klf5 and Klf6 mRNAs by ET-1 . Signalling through phosphoinositide 3 ′ kinase -LRB- PI3K -RRB- may increase expression of Klf2 -LSB- 36 -RSB- , and p38-MAPK is activated by ET-1 in cardiac myocytes -LSB- 37 -RSB- , so we examined the effects of 50 μM LY294002 or 5 μM SB203580 -LRB- selective inhibitors of PI3K and p38-MAPK , respectively -LSB- 34 -RSB- -RRB- . Surprisingly , LY294002 increased basal expression of Klf2 and Klf4 in cardiac myocytes and promoted further the increase in expression induced by ET-1 -LRB- Fig. 3B -RRB- , indicating that PI3K signalling is not required for the upregulation induced by ET-1 and suggesting that basal PI3K signalling negatively regulates Klf2/Klf4 expression . LY294002 did not affect Klf6 expression , but attenuated the increase in expression of Klf5 by ET-1 . Since basal PI3K signalling is required for protein synthesis in cardiac myocytes -LSB- 38 -RSB- , the effect of LY294002 on the increase in Klf5 induced by ET-1 -LRB- which requires de novo protein synthesis ; Fig. 2B -RRB- may be a reflection of this . SB203580 had a minimal effect on the basal expression of Klf2 , Klf4 , Klf5 or Klf6 , or on the increase in expression induced by ET-1 -LRB- Fig. 3B -RRB- . It is unlikely that p38-MAPK signalling plays a significant role in the response . 3.3 Translation-state analysis of Klf2 , Klf4 , Klf5 and Klf6 mRNA expression Given the increasing evidence for translational regulation of specific mRNAs -LSB- 39,40 -RSB- , we performed translation-state analysis of Klf2 , Klf4 , Klf5 or Klf6 mRNAs . We used qPCR to compare the relative levels of expression in the total RNA pool and in RNAs associated with cardiac myocyte polysomes -LRB- prepared by sucrose density centrifugation ; Fig. 4 , A and B -RRB- . Klf2 , Klf4 and Klf6 mRNAs were marginally enriched in cardiac myocyte polysomes in unstimulated cells and , following stimulation with ET-1 -LRB- 1 h -RRB- , the relative increase in the polysomes was at least as great as in the total RNA pool -LRB- Fig. 4 , C -- E -RRB- . Klf2 was increased 5.6-fold in the polysomes -LRB- 4.8-fold in total RNA -RRB- , Klf4 was increased 6.4-fold -LRB- 5.0-fold in total RNA -RRB- and Klf6 was increased 4.6-fold -LRB- 4.8-fold in total RNA -RRB- . Klf5 mRNA was marginally decreased in the polysomes relative to the total pool , but the increase in expression induced by ET-1 in the polysomes -LRB- 4.3-fold -RRB- was slightly higher than in the total pool -LRB- 3.9-fold -RRB- -LRB- Fig. 4F -RRB- . We therefore expect all these Klfs to be efficiently translated into protein . Consistent with this , Klf6 protein -LRB- detected as bands of ∼ 38 -- 40 kDa on Western blots -RRB- was increased in cardiac myocytes exposed to ET-1 , with maximal expression at ∼ 1 h -LRB- Fig. 4G -RRB- . It could be argued that early genes should be efficiently translated and this may be a property of all such genes . However , Irs2 was upregulated as an IEG by ET-1 -LRB- no inhibition by cycloheximide , Fig. 2A -RRB- , but the mRNA was not enriched in the polysomes and the increase in the polysomes was significantly less than that in the total RNA pool -LRB- Fig. 4H -RRB- . Similarly , for interleukin 1 receptor-like 1 -LRB- Il1rl1 -RRB- , a second phase gene -LRB- upregulation was inhibited by cycloheximide ; Fig. 2B -RRB- , the increase in expression in the polysomes was significantly less than in the total RNA pool -LRB- Fig. 4I -RRB- . It is therefore notable that Klf2 , Klf4 , Klf5 and Klf6 mRNAs are efficiently recruited to the polysomes for translation into protein . 3.4 Regulation of Klf2 , Klf4 and Klf6 by H2O2 and pro-inflammatory cytokines We considered whether other stimuli also regulate expression of Klf2 , Klf4 or Klf6 mRNAs . H2O2 -LRB- 0.2 mM -RRB- promotes cardiac myocyte apoptosis -LSB- 41 -RSB- , but also stimulates ERK1/2 -LRB- and other pathways -RRB- -LSB- 42 -RSB- , and stimulated a transient increase in expression of Klf2 -LRB- Fig. 5A -RRB- . However , the response was less than that of ET-1 -LRB- ∼ 3.6-fold -RRB- and was delayed -LRB- maximal increase at 1 h -RRB- -LRB- Fig. 1A -RRB- . Consistent with our previous report -LSB- 23 -RSB- , 0.2 mM H2O2 upregulated Klf4 and Klf6 mRNAs with maximal expression -LRB- 5.0-fold -RRB- from 1 h and sustained expression over at least 6 h -LRB- Fig. 5 , B and C -RRB- . We have observed a similar delay in induction of other IEGs by H2O2 compared with ET-1 -LRB- e.g. Fos family members , unpublished data -RRB- , which may reflect the delayed time course for activation of ERK1/2 signalling -LRB- maximal activation of ERK1/2 by ET-1 is at ∼ 5 min -LSB- 43 -RSB- ; maximal activation by H2O2 is at 15 -- 30 min -LSB- 42 -RSB- -RRB- . Consistent with a role for ERK1/2 signalling , U0126 inhibited the increase in expression of Klf2 , Klf4 and Klf6 by H2O2 -LRB- Fig. 5D -RRB- . As with ET-1 , SB203580 did not affect the response to H2O2 -LRB- Fig. 5D -RRB- , whereas LY294002 enhanced the response -LRB- Fig. 5E -RRB- . In endothelial cells , Klf2 is downregulated by TNFα -LSB- 15 -RSB- . IL-1β or TNFα profoundly and rapidly -LRB- from ∼ 1 h -RRB- downregulated Klf2 mRNA expression , a response which was sustained over at least 6 h -LRB- Fig. 6 , A and B -RRB- . Neither cytokine significantly affected the expression of Klf4 or Klf6 -LRB- data not shown -RRB- . The decrease in expression of Klf2 in response to IL-1β was not affected by either SB203580 or U0126 , although LY294002 -LRB- which itself increased expression of Klf2 mRNA , Fig. 3B -RRB- restored the baseline levels of expression -LRB- Fig. 6C -RRB- . 4 Discussion 4.1 Regulation of gene expression by Klf family members Many studies of individual Klfs relate to specific cells/tissues in which they are highly expressed or for which the phenotype of a transgenic mouse suggests they may play some role . Thus , most studies of Klf2 focus on its regulation in lung tissue , endothelial cells or T cells , whereas many studies of Klf4 are in gut , endothelial cells or smooth muscle cells , and Klf5 is studied largely in gut or smooth muscle cells . Although there are indications that Klfs are transcriptionally regulated in these various cells -LRB- e.g. Klf2 expression is increased by shear stress in endothelial cells -LSB- 14 -RSB- -RRB- , and there is an indication of interplay between Klf family members -LRB- e.g. Klf4 and Klf5 appear to operate in an antagonistic manner to regulate cell cycle progression -LSB- 8 -RSB- -RRB- , few studies consider the regulation and function of the Klfs as a family . Our data demonstrate substantial and dynamic regulation of nine Klfs by ET-1 in a single cell type -LRB- Fig. 1 -RRB- with at least three regulated as IEGs -LRB- Fig. 2A -RRB- , suggesting that Klfs potentially play a much more significant role in the phenotypic responses of more cell types than has so far been considered . Klfs are defined by the high degree of homology in their C-terminal DNA-binding domains -LSB- 1,6 -RSB- . It is probably not surprising , therefore , that all bind to similar consensus DNA sequences or , with 17 family members , that such elements are relatively common in gene promoters . The Klfs form two principal groups of transcriptional repressors -LRB- acting through CtBPs or mSin3a -RRB- plus a group which are primarily transcriptional activators , and it could be argued that the different Klf groups all do bind to similar sequences but the effect depends on whether the Klf is an activator or repressor and , if the latter , the mode of action . An additional factor could be that different cells express different Klfs and interactions with other cell-specific transcription factors may result in differential gene expression . In cardiac myocytes , we detected expression of at least 11 Klfs -LRB- Table 2 -RRB- , with regulation of several family members from each of the activator/repressor groups by ET-1 -LRB- Fig. 1 -RRB- , suggesting that -LRB- at least in this system -RRB- neither tissue specificity nor classification according to global function is responsible for functional differences between individual Klfs . Since there is evidence for functional specificity in other cells -LSB- 44,45 -RSB- , minor variations in primary and secondary structure may be sufficient for individual Klfs to have differing affinities for precise sequences in various gene promoters . Expression of any individual gene may therefore reflect the balance of Klfs expressed at any particular time in relation to the precise DNA-binding sites which can be accessed by them . 4.2 Regulation of expression of Klf2 , Klf4 , Klf5 and Klf6 Of the Klfs which were upregulated by ET-1 , Klf2 mRNA expression increased very rapidly and transiently -LRB- Fig. 1A -RRB- , and it was regulated as an IEG -LRB- Fig. 2A -RRB- . Partial inhibition of the response by U0126 -LRB- Fig. 3A -RRB- suggests that ERK1/2 signalling is required to some degree . ERK1/2 signalling was also required for upregulation of Klf2 by H2O2 -LRB- Fig. 5D -RRB- . In contrast to endothelial cells subjected to shear stress -LSB- 36 -RSB- , the increase in Klf2 expression by LY294002 and enhanced response to ET-1 in the presence of LY294002 -LRB- Fig. 3B -RRB- indicated that PI3K signalling negatively regulates Klf2 expression in cardiac myocytes . ERK5 phosphorylation of the transcription factor MEF2 promotes Klf2 expression in other cells -LSB- 46 -RSB- , but ERK5 is not significantly activated in cardiac myocytes by ET-1 -LSB- 24 -RSB- , ERK1/2 do not efficiently activate MEF2 -LSB- 47 -RSB- and inhibition of p38-MAPKs -LRB- which also phosphorylate MEF2 -LSB- 47 -RSB- -RRB- by SB203580 had no effect on Klf2 expression induced by ET-1 -LRB- Fig. 3B -RRB- , suggesting that MEF2 is not the principal factor involved in this context . Of the transcription factors associated with the Klf2 promoter identified by Ahmed and Lingrel -LSB- 48 -RSB- , the most probable candidate for regulating Klf2 expression by ET-1 in our study is CREB . CREB is rapidly -LRB- within 5 -- 10 min -RRB- phosphorylated in cardiac myocytes exposed to ET-1 , a response which is inhibited by U0126 -LSB- 49 -RSB- . Like Klf2 , the increases in expression of Klf4 and Klf6 -LRB- Fig. 1 , B and C -RRB- were inhibited by U0126 , -LRB- Figs. 3A and 5D -RRB- , and both were regulated as IEGs -LRB- Fig. 2A -RRB- . Our data are consistent with other systems in which Klf4 is regulated as an IEG by 15-deoxy-Δ12 ,14 prostaglandin J2 -LSB- 50 -RSB- or platelet-derived growth factor -LSB- 51 -RSB- , and Klf6 is regulated as an IEG by phorbol esters or serum -LSB- 52,53 -RSB- . However , for either Klf4 or Klf6 , the potential transcription factors or mRNA stabilisation factors which promote the increase in expression are not known . The increase in expression of Klf5 induced by ET-1 was inhibited by cycloheximide -LRB- Fig. 2B -RRB- , so the principal factors promoting its expression must be newly-synthesized proteins , and the response was inhibited by U0126 -LRB- Fig. 3A -RRB- , implicating ERK1/2 signalling in the response . These data are consistent with other studies in which phorbol esters or fibroblast growth factor increase Klf5 expression in smooth muscle cells in an ERK1/2-dependent manner -LSB- 54 -RSB- and , in fibroblasts , the increase in expression induced by phorbol esters is inhibited by cycloheximide -LSB- 55 -RSB- . Studies of mRNA expression raise the question of whether or not changes in mRNA expression equate to changes in protein expression . In cardiac myocytes treated with ET-1 , translation-state analysis indicated that mRNAs for Klf2 , Klf4 , Klf5 and Klf6 were increased to a similar or greater extent in the polysomes than in the total RNA pool -LRB- Fig. 4 -RRB- . Since ET-1 also increases the global rate of protein synthesis in cardiac myocytes -LSB- 56 -RSB- -LRB- illustrated by the increase in A254 profile of polysome fractions 6 -- 11 with concomitant decrease of monosome fractions 2 -- 4 ; Fig. 4A -RRB- , overall , we would predict that the rate of synthesis of each of the Klf proteins should be increased at least in proportion to the mRNA . Consistent with this , we detected a significant , transient increase in expression of Klf6 protein following stimulation with ET-1 -LRB- Fig. 4G -RRB- . Although cellular stresses such as oxidative stress can generally inhibit translation , 0.2 mM H2O2 does not have a significant effect on global protein synthesis over 4 h , and our microarray data indicate that Klf2 , Klf4 , and Klf6 are increased to a similar extent in the polysomes as in total RNA -LRB- data not shown -RRB- . 4.3 Regulating Klf-dependent gene expression in cardiac myocytes Downregulation of Klf15 is associated with the hypertrophic response of cardiac myocytes and may be required for hypertrophy to develop -LSB- 21 -RSB- . However , our data suggest that the situation regarding Klf-mediated regulation of gene expression in cardiac myocytes is more complex . In cardiac myocytes exposed to ET-1 , we detected a rapid decrease in expression of mRNAs for three established repressors of gene expression -LRB- Klf3 , Klf11 and Klf15 ; Fig. 1 , G , H and I -RRB- , with simultaneous rapid increases in expression of four Klfs more commonly associated with transcriptional activation -LRB- Klf2 , Klf4 , Klf5 and Klf6 ; Fig. 1 , A -- D -RRB- . In response to ET-1 , therefore , there appears to be an immediate overall switch towards increasing Klf-directed gene expression . However , the increases in expression of Klf2 , Klf4 , Klf5 and Klf6 , and decrease in expression of Klf3 were transient , with a delayed increase in expression of Klf9 and Klf10 , suggesting that the balance of Klf-regulated gene expression continues to change . A similar effect was detected with H2O2 , with an early increase in expression of Klf2 , Klf4 , Klf5 and Klf6 and simultaneous decrease in expression of Klf3 , Klf11 and Klf15 -LRB- Fig. 5 and -LSB- 23 -RSB- -RRB- . In contrast , Il-1β or TNFα promoted downregulation of Klf2 expression -LRB- Fig. 6 -RRB- with little effect on Klf4 or Klf6 . Clearly , the genes which each of the Klfs regulates in cardiac myocytes remain to be established .

O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O

cardiac myocytes
immediate early genes
gene expression
cytokines