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Postnatal survival of merkel cells depends on the low affinity neurotrophin receptor P75
ESDR I JSID I SID Abstracts
s154
0922
0919 CERAMIDE INDUCES APOPTOSIS IN A HUMAN KERATINOCYTE CELL LINE AND IS ACCENTUATED BY INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 3 Jane m John Lear. ‘7-P Gill. PVN Newcomb. CE Archer & Holly. Department of Denalology and ‘Division of Surgery, University of Bristol, Bristol Royal Infirmary Bnstol RS, RHW Previous work has shown that insulin-like gmwth factor binding protein 3 (IGFBP-3) marginally lnhibts cull growth but potent&& a physiological trigger of apoptosis via an IGF-indewndent mechanism in breast cancar eDRhelial cells (Gill ZP. Perks CM Newomb PVN & Holly JMP. IGFBP-3 predisposes breasicancer calls io programmed celi death in a non-IGF dependent manner. J B&I Chem IBB7: 272: 25302.7). Human keratinocyies can respond to but lack aulocdne expression of IGFs. Ceramide is a second messenger wthin the cell that in response to extracellular stimuli propagates the signal into the cell interiorto inlbate apnptosis. In this study a human keraiinocyte cell line (HaCaT) was used to investigate the effect of increasing doses of ceramide with 0, without the addition of IGFBP-3 in inducing apoptosis. The cell permeable ceramide analague. C2 was used to induce apaptosis in the HaCaT cdls. Apoptosis was established using metabolic assay. trypan blue uptake. flow cytometry and cell cycle analysis. An increase in pre-Gl cells in the cell cycle, as measured by flow cylomeby, was interpreted as being indlcatlve of apoptosis. Incubation of HaCaT cells with C2 (1-1001rM)resulted in dose-dependent cell death by apoptosis. With increasing 4050s of C2 up tolOOph4 bypan Mue counts showed increasing cell death and flow cyiomeby showed pm-01 cdls increasing from 2-3x control levels (p
POSTNATAL SURVIVAL OF MERKEL CELLS DEPENDS ON THE LOW AFFINITY NEUROTROPHIN RECEPTOR P75. I. Bemmann. C. L. Stuckv’. K. V. Tovka’and M. Kolkenbum’. Department of Dannatdlcgy and Neur&2y* Wtirrburg, Germany. Merkel cells are the end organs of slowly adapting low thrashold ma&andmcepton (SA fibers). Hera. wa analysed the posk?atal development of M&al calls in the skin of miae with lack the laafftnii neumtmphkt receptor ~75. To count the number of M&al cells in the touch dome complexas of bank skin the vital dye quinscrine was used. In wtidtype mice (WT) there was a small but significant loss in the number nf Merkel calls per cm2 fmm 2 weeks to 2 months, but no further loss at 6 months. However, in ~754 mica theta was a sawn loss in the number of Merkel calls from 2 waaks to 2 months, but no further loss at 6 months. This raductiin was accounted for by a loss of Me number of toudh domes par area of back skin rather than by a radudion of the number of Merkal cells per dome. We also axamined Merkel calls in glabrous skin of the foot pad by immunoftuurascanca using an antibody against cytokerattn 15 (CK15). In WT mice the numbar uf Marks4 calls remained stable fmm 2 waeks to 2 months and to 6 months. In ~754 mice there was a gradual decrease in the number of Markal calls par mm epldennis from 2 waaks to 2 months and to 8 months. To assess wther Me SA Rben innarvating Mekel dells were &fasted by the loss of Merkel calls standard etastrophysidDgical techniques wara used to anatyss the ralativs number and functtonat pmper5es cd the SA fibers. We found no loss in the number of SA fibers in the ~754 at 2 weeks, 2 rwnths or 6 months. Furthemtore. the medtaniil raspcmse properties of SA fibers in ~75/- mica at tha three time points wara not dKfwent fmm SA 5bres in adult WT mice. Thus. our data indicate that tlw postnatal sur.4val of Merkal cells Is dependant on tha prasanna of p75 and that neKber the sun&at nor the function of SA fibars is impeded by the loss of Merkel cells.
0920
0923
NEUROPEPTIDE-CONTAINit.lG GFlBERS AND THE WOUND HEALING PROCEBS IN THE RAT SKIN; NEITHER CAPBAICIN NOR PERIPHERAL NEUROTDMY AFFECTS THE RATE OF HEALING. Joanna Wallernl~ka(l), Duan Chen (2). Frank Sundler_(3).Depa1tmenls cd Dermaldogy (1). Pharmacology (2) ard Physiology and Neurosdenx, (3). Lund Universily. B- 22185 Ltnd. Sweden. Wound healing in rat sldn was studied in standardiied wounds inRict6-dafter s&tic nerve sectioning andlor cxpsaioin-induced depletion of sensory nerve (C-fiber) newopeptids content. Healing of wounds inflicted on the proximal medial aspect d both hind legs was studied in 30 rats pretreated tih capsaicin. as compared with SO untreated rats. Healing of wounds inflicted on lhs proximal lateral asp+ct of both hind legs was stud!ed in 40 rats undergoing unilateral e&tic new sectioning, the contralateral hind legs saving as controls. Fiieen capsaiziMeated rats also underwent unilateral sciatic nave sectioning, the contralateml hind legs again sewing as ccMols. Wound healing via6 assessedby daily visual inqwction. histdqic examination, and antibodies against substance P (SP), calckonin gene&ted pepltde (CGRP), vasoactiie inteStiMl peptide (VIP), neuropeptide Y CPY) and a panneuronal marker, prdein gene product 9.5 (PGP), were used to determine the distribution of &ma1 nowe fibers In contrds nerve fiber density in the wound tissue was low during the firs( few days after wand infliction. but started to illcrease on day 4. reaching a peak on day 7 when 25 % d medial wounds end 70% of lateral wands were healed. All wounds were healed on day 11, a scar appearing on day 14 followed by a deaease in nave fiber density. Capsaicin treatment andbr sciatic nerve sectioning reduced the density of CGRP immuncfeacttie new fibers by 70% and that of PGP-immuweactive fibas by 50%. The caps&in-induced reduction in PGPimmuncreactive nerve fiber density is atlrbutable to partial destruction d pwpheral ,wwe tin. CGRPand PGP-immunoreactiie nave fiber density was mstcwd both in capsaicin treated and denewated groups, reachinga maximum. correspondingtolhe original Iwel. by days4-10. Neither the reduction in new fiber de&y fdbwing sciatic nerve sectioning no, the impirrrmnt of sensory nave functional capacity following caps&n treatment affected Ihe rate of wound healing. all wounds being closed on day 11.
STRETCHING OF HACAT CELLS INDUCES SAPK ACTIVATION AND MAPK DE-ACTIVATION. m. A Band M Gwchel. R. m Department of Demuadogy, Univemity of Fmnkliui ‘Medical!&ml, Fra&iw?&n, Germany Human skin is permanently exposed to mechanical stretching. In contrast the common culture technicluw for cell nrpansion miss this prombwtt featwe. In order to study whether mechanical stretching plays a role for human kemtincqtes we sstsblish s culture technique which atlows to mimic this physical signst: HaCaT c&Is were cultivated on silicon suppotts which underxo _ s cyclic stnstchirut of about 10% of the
’
’ JMP
____. ,_IK
cyiometry
Activation of SAPK (stress activated protein kbwe) was daected by immunoprecipitation with ohm fusion protein beads. Later in vitro phosnhowtatiort was pertbrrn&t &d phosphn-c-hut visu&d by cotwcutivs wastam ho&. -The active form of MAPK (mitogen aaivated protein kinase) was immuaoprecipitated with a phospho-MAPK antibody. Later in vitro phosphwylation of tmnsaiption factor Elk 1 was performed. By western blotig the phosphmyiated Elk 1 was detected usiq a phosph+Elk 1 antibody. Ah Smbtof cyclic stretching the mechrtnicalforce ~89 stopped. It was fomtd that between 30 and 60 min the SAPK wtititv bweased whereas MAPK activitv dmeased SAPK actiwtioo is w&y described f&r various cxtmcolhdar stimoli in&ding Wlight, heat shock oxidant stress and osmotic stress, rwpectively. It is likely that SAPK activation and the conwutive activation oftmn&ption factor AP-1 is part of a general response to environmmrtal stress. Our findings suggest that me&&-al stretching of HaCaT alIs stimulstes activation of stress response elements and suppresses the protein cascade mediating the proliferation signal.
0924
0921 CHAP.ACTERtZATlON
initial length.
OF NEWBORN SKIN AND ADULT
SKIN GRAFTS
FROM
TGF-!3 2 KNOCK-OUT MICE. Kerstin’_&XF%uzPausZ.2HandiisF;1. poetschman~ and G, Paola DQ&-, Cutaneous Biology Research Center, Massachusetts General Hospital / Harvard, Charlestown, MA, USA, 2 Department of
Dermatology, Charit& Humboldt-Universitit N Berlin, Germany, 3 Departments of Molecular Genetics, Biochemistry and Microbiology, Unwersity of Cmcinnati. Cincinnati, USA. Members of the TGF-0 family are potent regulators of cell growth and differentiation. The distinct expressmn of the three TGF-0, isoforms m the skin suggests different activities in development and adult homeostasis. To elucidate these still elusive functions as they relate specifically to TGF-0 2, we have characterized several pammcters of keratinacyte probferation and differentiation of mice wth a bomozygous disruption of the TGF-8 2 gene. These mice die at bnth because of developmental abnormalities wblcb do overlap with those of TGF-0.t and 3 null mice. Initially, we have characterized the backskin of newborn TGF-.2 knack-out mice far several parameters of differentiation (Kemtin 1, Keratin 5, tilaggrin, etc.) and proliferation (BrdU labelling, Ki-67), by immunohistology of cryosections. To invesugate the consequence of lack of TGF-B.2 in adult skin, we have grafted the skin of newborn TGF-l3.2knock-outmice onto nude mice. At one month after grafting, the grafted knockout skins appear grossly normal, indicating that TGF-B 2 function 1s probably not essential for development and maturation of the skin even after birth. Experiments will be presented which further test this powbihty and assess whether TGF-0.2 function 1s required under conditions of altered tissue hamoeostasis such as during wound healmg.
not
BLOCKING INHIBITS B. Jam&,
OF RAS FUNCTION HUMAN
MELANOMA
BY A COMPETITIVE
RAS
ANTAGONIST
GROWTH
H. Schlagbauer-Wadl’z, H. Kah?, E. Ress I’, B. May@, H.-G. Eichle8, Y. KIoog’, H. Pehambxger’, and K. Walt?. Department of Dermatology, Division of General Dermatology’, and Department of Clinical Pharmacology, Section of Experimental Oncolo&, University of Vienna, Viemm, Austria, and Department of Neurobiochemishy3, Tel Aviv University, Tel Aviv, Israel. During the past decades, knowledge of melanoma biology has increased considerably and munerous therapeutic modalities me currently under investigation. Advanced melmvxna nevertheless remains a prime example for poor treatment response and the search for new treatment concepts is clearly warranted. Ras gene products play a key role in tyrosine kinase growth factor signaling thought to be generally of impatance also in oncogenesis and tumor progression. Therefore, it is reasonable to speculate that a pharmacological approach curtailing ms activity may be capable of inhibiting tumor growth Testing this concept, we evaluated the anti-tumor activity of S-tram, tmnsfamesylthiosalicylic acid (FTS), a recently discovered competitive inhibitor of ms signal tmosduction that dislodges ms from its membrane anchoring site, in vitro and in two independent SCID mouse xenotmnsplantation models of human melanoma expressing either wild-type ras or activated ms. A statistically significant reduction of melanoma cell growth in vitro as well as of melanoma growth in viva could be observed in both systems without evidence of FTS related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors, that either carry activated ras genes OI rely more heavily ras signal transduction than non-malignant cells.
on
s154
0922
0919 CERAMIDE INDUCES APOPTOSIS IN A HUMAN KERATINOCYTE CELL LINE AND IS ACCENTUATED BY INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 3 Jane m John Lear. ‘7-P Gill. PVN Newcomb. CE Archer & Holly. Department of Denalology and ‘Division of Surgery, University of Bristol, Bristol Royal Infirmary Bnstol RS, RHW Previous work has shown that insulin-like gmwth factor binding protein 3 (IGFBP-3) marginally lnhibts cull growth but potent&& a physiological trigger of apoptosis via an IGF-indewndent mechanism in breast cancar eDRhelial cells (Gill ZP. Perks CM Newomb PVN & Holly JMP. IGFBP-3 predisposes breasicancer calls io programmed celi death in a non-IGF dependent manner. J B&I Chem IBB7: 272: 25302.7). Human keratinocyies can respond to but lack aulocdne expression of IGFs. Ceramide is a second messenger wthin the cell that in response to extracellular stimuli propagates the signal into the cell interiorto inlbate apnptosis. In this study a human keraiinocyte cell line (HaCaT) was used to investigate the effect of increasing doses of ceramide with 0, without the addition of IGFBP-3 in inducing apoptosis. The cell permeable ceramide analague. C2 was used to induce apaptosis in the HaCaT cdls. Apoptosis was established using metabolic assay. trypan blue uptake. flow cytometry and cell cycle analysis. An increase in pre-Gl cells in the cell cycle, as measured by flow cylomeby, was interpreted as being indlcatlve of apoptosis. Incubation of HaCaT cells with C2 (1-1001rM)resulted in dose-dependent cell death by apoptosis. With increasing 4050s of C2 up tolOOph4 bypan Mue counts showed increasing cell death and flow cyiomeby showed pm-01 cdls increasing from 2-3x control levels (p
POSTNATAL SURVIVAL OF MERKEL CELLS DEPENDS ON THE LOW AFFINITY NEUROTROPHIN RECEPTOR P75. I. Bemmann. C. L. Stuckv’. K. V. Tovka’and M. Kolkenbum’. Department of Dannatdlcgy and Neur&2y* Wtirrburg, Germany. Merkel cells are the end organs of slowly adapting low thrashold ma&andmcepton (SA fibers). Hera. wa analysed the posk?atal development of M&al calls in the skin of miae with lack the laafftnii neumtmphkt receptor ~75. To count the number of M&al cells in the touch dome complexas of bank skin the vital dye quinscrine was used. In wtidtype mice (WT) there was a small but significant loss in the number nf Merkel calls per cm2 fmm 2 weeks to 2 months, but no further loss at 6 months. However, in ~754 mica theta was a sawn loss in the number of Merkel calls from 2 waaks to 2 months, but no further loss at 6 months. This raductiin was accounted for by a loss of Me number of toudh domes par area of back skin rather than by a radudion of the number of Merkal cells per dome. We also axamined Merkel calls in glabrous skin of the foot pad by immunoftuurascanca using an antibody against cytokerattn 15 (CK15). In WT mice the numbar uf Marks4 calls remained stable fmm 2 waeks to 2 months and to 6 months. In ~754 mice there was a gradual decrease in the number of Markal calls par mm epldennis from 2 waaks to 2 months and to 8 months. To assess wther Me SA Rben innarvating Mekel dells were &fasted by the loss of Merkel calls standard etastrophysidDgical techniques wara used to anatyss the ralativs number and functtonat pmper5es cd the SA fibers. We found no loss in the number of SA fibers in the ~754 at 2 weeks, 2 rwnths or 6 months. Furthemtore. the medtaniil raspcmse properties of SA fibers in ~75/- mica at tha three time points wara not dKfwent fmm SA 5bres in adult WT mice. Thus. our data indicate that tlw postnatal sur.4val of Merkal cells Is dependant on tha prasanna of p75 and that neKber the sun&at nor the function of SA fibars is impeded by the loss of Merkel cells.
0920
0923
NEUROPEPTIDE-CONTAINit.lG GFlBERS AND THE WOUND HEALING PROCEBS IN THE RAT SKIN; NEITHER CAPBAICIN NOR PERIPHERAL NEUROTDMY AFFECTS THE RATE OF HEALING. Joanna Wallernl~ka(l), Duan Chen (2). Frank Sundler_(3).Depa1tmenls cd Dermaldogy (1). Pharmacology (2) ard Physiology and Neurosdenx, (3). Lund Universily. B- 22185 Ltnd. Sweden. Wound healing in rat sldn was studied in standardiied wounds inRict6-dafter s&tic nerve sectioning andlor cxpsaioin-induced depletion of sensory nerve (C-fiber) newopeptids content. Healing of wounds inflicted on the proximal medial aspect d both hind legs was studied in 30 rats pretreated tih capsaicin. as compared with SO untreated rats. Healing of wounds inflicted on lhs proximal lateral asp+ct of both hind legs was stud!ed in 40 rats undergoing unilateral e&tic new sectioning, the contralateral hind legs saving as controls. Fiieen capsaiziMeated rats also underwent unilateral sciatic nave sectioning, the contralateml hind legs again sewing as ccMols. Wound healing via6 assessedby daily visual inqwction. histdqic examination, and antibodies against substance P (SP), calckonin gene&ted pepltde (CGRP), vasoactiie inteStiMl peptide (VIP), neuropeptide Y CPY) and a panneuronal marker, prdein gene product 9.5 (PGP), were used to determine the distribution of &ma1 nowe fibers In contrds nerve fiber density in the wound tissue was low during the firs( few days after wand infliction. but started to illcrease on day 4. reaching a peak on day 7 when 25 % d medial wounds end 70% of lateral wands were healed. All wounds were healed on day 11, a scar appearing on day 14 followed by a deaease in nave fiber density. Capsaicin treatment andbr sciatic nerve sectioning reduced the density of CGRP immuncfeacttie new fibers by 70% and that of PGP-immuweactive fibas by 50%. The caps&in-induced reduction in PGPimmuncreactive nerve fiber density is atlrbutable to partial destruction d pwpheral ,wwe tin. CGRPand PGP-immunoreactiie nave fiber density was mstcwd both in capsaicin treated and denewated groups, reachinga maximum. correspondingtolhe original Iwel. by days4-10. Neither the reduction in new fiber de&y fdbwing sciatic nerve sectioning no, the impirrrmnt of sensory nave functional capacity following caps&n treatment affected Ihe rate of wound healing. all wounds being closed on day 11.
STRETCHING OF HACAT CELLS INDUCES SAPK ACTIVATION AND MAPK DE-ACTIVATION. m. A Band M Gwchel. R. m Department of Demuadogy, Univemity of Fmnkliui ‘Medical!&ml, Fra&iw?&n, Germany Human skin is permanently exposed to mechanical stretching. In contrast the common culture technicluw for cell nrpansion miss this prombwtt featwe. In order to study whether mechanical stretching plays a role for human kemtincqtes we sstsblish s culture technique which atlows to mimic this physical signst: HaCaT c&Is were cultivated on silicon suppotts which underxo _ s cyclic stnstchirut of about 10% of the
’
’ JMP
____. ,_IK
cyiometry
Activation of SAPK (stress activated protein kbwe) was daected by immunoprecipitation with ohm fusion protein beads. Later in vitro phosnhowtatiort was pertbrrn&t &d phosphn-c-hut visu&d by cotwcutivs wastam ho&. -The active form of MAPK (mitogen aaivated protein kinase) was immuaoprecipitated with a phospho-MAPK antibody. Later in vitro phosphwylation of tmnsaiption factor Elk 1 was performed. By western blotig the phosphmyiated Elk 1 was detected usiq a phosph+Elk 1 antibody. Ah Smbtof cyclic stretching the mechrtnicalforce ~89 stopped. It was fomtd that between 30 and 60 min the SAPK wtititv bweased whereas MAPK activitv dmeased SAPK actiwtioo is w&y described f&r various cxtmcolhdar stimoli in&ding Wlight, heat shock oxidant stress and osmotic stress, rwpectively. It is likely that SAPK activation and the conwutive activation oftmn&ption factor AP-1 is part of a general response to environmmrtal stress. Our findings suggest that me&&-al stretching of HaCaT alIs stimulstes activation of stress response elements and suppresses the protein cascade mediating the proliferation signal.
0924
0921 CHAP.ACTERtZATlON
initial length.
OF NEWBORN SKIN AND ADULT
SKIN GRAFTS
FROM
TGF-!3 2 KNOCK-OUT MICE. Kerstin’_&XF%uzPausZ.2HandiisF;1. poetschman~ and G, Paola DQ&-, Cutaneous Biology Research Center, Massachusetts General Hospital / Harvard, Charlestown, MA, USA, 2 Department of
Dermatology, Charit& Humboldt-Universitit N Berlin, Germany, 3 Departments of Molecular Genetics, Biochemistry and Microbiology, Unwersity of Cmcinnati. Cincinnati, USA. Members of the TGF-0 family are potent regulators of cell growth and differentiation. The distinct expressmn of the three TGF-0, isoforms m the skin suggests different activities in development and adult homeostasis. To elucidate these still elusive functions as they relate specifically to TGF-0 2, we have characterized several pammcters of keratinacyte probferation and differentiation of mice wth a bomozygous disruption of the TGF-8 2 gene. These mice die at bnth because of developmental abnormalities wblcb do overlap with those of TGF-0.t and 3 null mice. Initially, we have characterized the backskin of newborn TGF-.2 knack-out mice far several parameters of differentiation (Kemtin 1, Keratin 5, tilaggrin, etc.) and proliferation (BrdU labelling, Ki-67), by immunohistology of cryosections. To invesugate the consequence of lack of TGF-B.2 in adult skin, we have grafted the skin of newborn TGF-l3.2knock-outmice onto nude mice. At one month after grafting, the grafted knockout skins appear grossly normal, indicating that TGF-B 2 function 1s probably not essential for development and maturation of the skin even after birth. Experiments will be presented which further test this powbihty and assess whether TGF-0.2 function 1s required under conditions of altered tissue hamoeostasis such as during wound healmg.
not
BLOCKING INHIBITS B. Jam&,
OF RAS FUNCTION HUMAN
MELANOMA
BY A COMPETITIVE
RAS
ANTAGONIST
GROWTH
H. Schlagbauer-Wadl’z, H. Kah?, E. Ress I’, B. May@, H.-G. Eichle8, Y. KIoog’, H. Pehambxger’, and K. Walt?. Department of Dermatology, Division of General Dermatology’, and Department of Clinical Pharmacology, Section of Experimental Oncolo&, University of Vienna, Viemm, Austria, and Department of Neurobiochemishy3, Tel Aviv University, Tel Aviv, Israel. During the past decades, knowledge of melanoma biology has increased considerably and munerous therapeutic modalities me currently under investigation. Advanced melmvxna nevertheless remains a prime example for poor treatment response and the search for new treatment concepts is clearly warranted. Ras gene products play a key role in tyrosine kinase growth factor signaling thought to be generally of impatance also in oncogenesis and tumor progression. Therefore, it is reasonable to speculate that a pharmacological approach curtailing ms activity may be capable of inhibiting tumor growth Testing this concept, we evaluated the anti-tumor activity of S-tram, tmnsfamesylthiosalicylic acid (FTS), a recently discovered competitive inhibitor of ms signal tmosduction that dislodges ms from its membrane anchoring site, in vitro and in two independent SCID mouse xenotmnsplantation models of human melanoma expressing either wild-type ras or activated ms. A statistically significant reduction of melanoma cell growth in vitro as well as of melanoma growth in viva could be observed in both systems without evidence of FTS related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors, that either carry activated ras genes OI rely more heavily ras signal transduction than non-malignant cells.
on