Heat shock protein 25 plays multiple roles during mouse skin development

Heat shock protein (Hsp) 25 is a member of the small Hsp family. High levels of Hsp25 can be detected in skin. During adult epidermis differentiation, the concentration of Hsp25 increases as the distance of keratinocytes from the basal layer increases, in parallel with the extent of keratinization. We previously showed that Hsp25, mouse keratin (MK) 5, and MK14 participated in the formation of characteristic ring-shaped aggregates during the differentiation of the PAM212 keratinocyte cell line. We suggested that Hsp25 was involved in the disorganization of the MK5-MK14 keratin network before the establishment of the MK1-MK10 keratin network at the beginning of epidermis stratification. In this study, we have investigated the distribution of Hsp25 and keratins throughout skin development. We demonstrate that the distribution of Hsp25 and MK5 in the epidermis at the beginning of stratification and before keratinization is similar to that observed in PAM212 keratinocytes. These results indicate that there is a strong correlation between the mechanism we described ex vivo and the events taking place in vivo. Moreover, we show that Hsp25 is produced in different cell types in the epidermis and in the hair follicle at different stages of their development. Thus, our results suggest that Hsp25 is involved in more than one process during skin development.     

The expression and modulation of IL-1 alpha in murine keratinocytes

Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low [Ca2+] tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high [Ca2+] media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low [Ca2+] conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes. Thus LPS, UV, and cell differentiation state have a significant effect on expression of IL-1 alpha in murine keratinocytes.     

Characterization of a keratinocyte-derived T cell growth factor distinct from interleukin 2 and B cell stimulatory factor 1

Epidermal epithelial cells (keratinocytes) produce and secrete a variety of immunologically active cytokines. We have previously reported that both transformed (PAM 212) and normal murine keratinocytes produce a soluble factor which induces proliferation of the T cell line, HT-2. In the present study we sought to compare keratinocyte-derived T cell growth factor (KTGF) with other T cell growth factors, characterize its physicochemical properties, and substantially purify KTGF from PAM 212 conditioned medium. KTGF from PAM 212 conditioned medium was not inhibited by antibodies which block the effect of interleukin 2 (IL 2) (S4B6) or B cell stimulatory factor 1 (BSF 1) (11B11). KTGF is heat-stable, has an isoelectric point of 4.8, and a relative molecular mass of 16 to 23 kilodaltons under nonreducing conditions. KTGF activity was enhanced at least 41,413-fold by sequential hydroxylapatite bulk preparation, desalting by reversed-phase chromatography, gel filtration high pressure liquid chromatography (HPLC), and reversed-phase HPLC. Keratinocytes produce a T cell growth factor with physicochemical properties distinct from IL 2 and BSF 1. KTGF may play a role in regulating the growth and differentiation of T cells in the epidermis.     

Hsp25 and the p38 MAPK pathway are involved in differentiation of cardiomyocytes

The small heat-shock protein HSP25 is expressed in the heart early during development, and although multiple roles for HSP25 have been proposed, its specific role during development and differentiation is not known. P19 is an embryonal carcinoma cell line which can be induced to differentiate in vitro into either cardiomyocytes or neurons. We have used P19 to examine the role of HSP25 in differentiation. We found that HSP25 expression is strongly increased in P19 cardiomyocytes. Antisense HSP25 expression reduced the extent of cardiomyocyte differentiation and resulted in reduced expression of cardiac actin and the intermediate filament desmin and reduced level of cardiac mRNAs. Thus, HSP25 is necessary for differentiation of P19 into cardiomyocytes. In contrast, P19 neurons did not express HSP25 and antisense HSP25 expression had no effect on neuronal differentiation. The phosphorylation of HSP25 by the p38/SAPK2 pathway is known to be important for certain of its functions. Inhibition of this pathway by the specific inhibitor SB203580 prevented cardiomyocyte differentiation of P19 cells. In contrast, PD90589, which inhibits the ERK1/2 pathway, had no effect. Surprisingly, cardiogenesis was only sensitive to SB203580 during the first 2 days of differentiation, before HSP25 expression increases. In contrast to the effect of antisense HSP25, SB203580 reduced the level of expression of the mesodermal marker Brachyury-T during differentiation. Therefore, we propose that the p38 pathway acts on an essential target during early cardiogenesis. Once this initial step is complete, HSP25 is necessary for the functional differentiation of P19 cardiomyocytes, but its phosphorylation by p38/SAPK2 is not required.     

Macrophage colony-stimulating factor production by murine and human keratinocytes. Enhancement by bacterial lipopolysaccharide

CSF have a broad range of effects on differentiated cells outside the bone marrow. Site-specific elaboration of these factors may influence local immune reactions. Keratinocytes have been demonstrated to produce a number of immunoactive cytokines, including factors capable of modifying macrophage function. We have previously identified at least two products of keratinocytes that induce DNA synthesis by elicited peritoneal macrophages; one factor has been identified as granulocyte-macrophage CSF. In the present study, the second keratinocyte product has been characterized and identified as macrophage-CSF (M-CSF). Conditioned media from cultures of normal human keratinocytes and the transformed murine keratinocyte cell line PAM 212 induce formation of macrophage colonies in soft agar as well as dose-dependent proliferation of the M-CSF-dependent cell line BAC1.2F5. The bioactivity in both assays is blocked by neutralizing anti-M-CSF antibody. Western blot analysis of cell lysates from both PAM 212 and normal human keratinocytes demonstrates multiple molecular mass forms of M-CSF (45 to 98 kDa). Northern blot analysis (PAM 212 cells) and in situ hybridization (normal keratinocytes) demonstrate expression of M-CSF mRNA. Stimulation of keratinocytes with LPS increases M-CSF synthesis as measured both by bioactivity and level of mRNA expression. Thus, both murine and human keratinocytes produce M-CSF in vitro. Furthermore, production of keratinocyte-derived M-CSF is increased by bacterial LPS. CSF production by keratinocytes may play an important role in regulating the cutaneous immune response.     

The small heat shock protein hsp25 is accumulated in P19 embryonal carcinoma cells and embryonic stem cells of line BLC6 during differentiation

Murine embryonal carcinoma and embryonic stem cell lines were investigated with regard to the occurrence of the small heat shock protein hsp25 during cell growth and differentiation. In the embryonal carcinoma cell line F9 considerable constitutive levels of hsp25 were observed which could be slightly increased by treatment with retinoic acid. No hsp25 was found, however, in the embryonal carcinoma cell line PCC4. When analyzing the pluripotent embryonal carcinoma cell line P19 and the pluripotent embryonic stem cell line BLC6, both characterized by high differentiation capacity, no hsp25 was observed under cell culture conditions maintaining the undifferentiated state. Induction of differentiation caused by prolonged cell culture, retinoic acid treatment, or embryoid body formation, however, resulted in an increase of the level of hsp25. The finding that hsp25 is accumulated in a differentiation-dependent manner suggests that this protein is associated with processes involved in differentiation. Therefore, hsp25 can be regarded as a marker of differentiation in the investigated embryonal carcinoma cell line P19 and the embryonic stem cell line BLC6.     

Overexpression of murine small heat shock protein HSP25 interferes with chondrocyte differentiation and decreases cell adhesion

Although multiple functions for the small heat shock protein HSP25 have been proposed, its specific role during developmental and differentiation processes is not known. Cartilage is one of the tissues in which HSP25 is specifically and highly expressed during development. C1 cells, able to form aggregates in vitro, can be induced to differentiate into chondrocytes. In this study, we generated two stable transfected clones overexpressing HSP25 at two different levels. Cell morphology and growth rate were modified in both clones, although the actin content and distribution did not seem to be altered. Overexpressing clones had more difficulties in coalescing, leading to smaller aggregates and they did not differentiate into chondrocytes. Subsequently, these aggregates tended to dissociate into loose masses of dying cells. The strength of all these effects was directly correlated to the level of HSP25 overexpression. These data suggest that overexpressing HSP25 decreases cellular adhesion and interferes with chondrocyte differentiation.     

Over-Expression of HSP47 Augments Mouse Embryonic Stem Cell Smooth Muscle Differentiation and Chemotaxis

In the recent decade, embryonic stem cells (ESC) have emerged as an attractive cell source of smooth muscle cells (SMC) for vascular tissue engineering owing to their unlimited self-renewal and differentiation capacities. Despite their promise in therapy, their efficacy is still hampered by the lack of definitive SMC differentiation mechanisms and difficulties in successful trafficking of the ESC towards a site of injury or target tissue. Heat shock protein 47 (HSP47) is a 47-kDa molecular chaperone that is required for the maturation of various types of collagen and has been shown to be a critical modulator of different pathological and physiological processes. To date, the role of HSP47 on ESC to SMC differentiation or ESC chemotaxis is not known and may represent a potential molecular approach by which ESC can be manipulated to increase their efficacy in clinic. We provide evidence that HSP47 is highly expressed during ESC differentiation into the SMC lineage and that HSP47 reduction results in an attenuation of the differentiation. Our experiments using a HSP47 plasmid transfection system show that gene over-expression is sufficient to induce ESC-SMC differentiation, even in the absence of exogenous stimuli. Furthermore, HSP47 over-expression in ESC also increases their chemotaxis and migratory responses towards a panel of chemokines, likely via the upregulation of chemokine receptors. Our findings provide direct evidence of induced ESC migration and differentiation into SMC via the over-expression of HSP47, thus identifying a novel approach of molecular manipulation that can potentially be exploited to improve stem cell therapy for vascular repair and regeneration.     

PAM14, a novel MRG- and Rb-associated protein, is not required for development and T-cell function in mice

PAM14 has been found to associate in complexes with the MORF4/MRG family of proteins as well as Rb, the tumor suppressor protein. This suggested that it might be involved in cell growth, immortalization, and/or senescence. To elucidate the in vivo function of PAM14, we characterized the expression pattern of mouse Pam14 and generated PAM14-deficient (Pam14(-/-)) mice. Pam14 was widely expressed in all mouse tissues and as early as 7 days during embryonic development. Despite this ubiquitous expression in wild-type mice, Pam14(-/-) mice were healthy and fertile. Response to mitogenic stimulation and production of interleukin-2 were the same in stimulated splenic T cells from Pam14(-/-) mice as in control littermates. Cell growth rates of mouse embryonic fibroblasts (MEFs) from all three genotypes were the same, and immortalized cells were obtained from all cell cultures during continuous culture. There was also no difference in expression of growth-related genes in response to serum stimulation in the null versus control MEFs. These data demonstrate that PAM14 is not essential for normal mouse development and cell cycle control. PAM14 likely acts as an adaptor protein in nucleoprotein complexes and is probably compensated for by another functionally redundant protein(s).     

Keratinocyte-derived granulocyte/macrophage colony-stimulating factor induces DNA synthesis by peritoneal macrophages

Keratinocytes have been demonstrated to produce a number of cytokines, including growth factors such as the CSF IL-3. Circulating blood monocytes and some elicited macrophages retain a significant proliferative potential in response to colony-stimulating activity. Because a macrophage response is prominent in a variety of cutaneous immune reactions, we have studied the ability of conditioned media (CM) from a transformed murine keratinocyte cell line (PAM 212) and from normal murine keratinocytes to induce growth of peritoneal macrophages. CM from both normal and transformed keratinocyte cultures induces [3H]thymidine incorporation by thioglycollate-elicited, but not resident, peritoneal macrophages. IEF of PAM 212 CM reveals peaks of activity at pI 4.8 and less than or equal to 4.2. Analysis of CM by reversed-phase HPLC demonstrates active fractions that elute at 46 to 48% and 53 to 55% acetonitrile. The Mr of the 46 to 48% acetonitrile factor is 25 to 30 kDa by gel filtration HPLC. Polyclonal anti-granulocyte/macrophage (GM) CSF antibody blocks the induction of macrophage [3H]thymidine incorporation by factors with pI 4.8 and eluting at 46 to 48% acetonitrile but does not reduce the activity of crude CM or the factor eluting at 53 to 55% acetonitrile. Based on both physiochemical criteria and antibody neutralization, keratinocytes produce GM-CSF. Keratinocyte-derived factors, including GM-CSF, may play an important role in regulating cutaneous macrophage responses.     

Function of 90-kDa heat shock protein in cellular differentiation of human embryonal carcinoma cells

Summary Heat shock proteins (HSPs) have been recognized as molecules that maintain cellular homeostasis during changes in the environment. Here we report that HSP90 functions not only in stress responses but also in certain aspects of cellular differentiation. We found that HSP90 slowed remarkably high expression in undifferentiated human embryonal carcinoma (EC) cells, which were subsequently dramatically down-regulated during in vitro cellular differentiation, following retinoic acid (RA) treatment, at the protein level. Surprisingly, heat shock treatment also triggered the down-regulation of HSP90 within 48 h at the protein level. Furthermore, the heat treatment induced cellular differentiation into neural cells. This down-regulation of HSP90 by heat treatment was shifted to an up-regulation attern after cellular differentiation in response to RA treatment. In order to clarify the functions of HSP90 in cellular differentiation, we conducted various experiments, including overexpression of HSP90 via gene transfer. We showed that the RA-induced differentiation of EC cells into a neural cell lineage was inhibited by overexpression of the HSP90α or-β isoform via the gene transfer method. On the other hand, the overexpression of HSP90β alone impaired cellular differentiation into trophoectoderm. These results show that down-regulation of HSP90 is a physiological critical event in the differentiation of human EC cells and that specific HSP90 isoforms may be involved in differentiation into specific cell lineages.     

Comparative proteomic analysis of differentiation of mouse F9 embryonic carcinoma cells induced by retinoic acid

The multipotent mouse F9 embryonic carcinoma cell is an ideal model system to investigate the mechanism of retinoic acid (RA) in cell differentiation and cell growth control and the biochemical basis of early embryonic development. We reported here a proteomics approach to study protein expression changes during the differentiation of F9 cells into the visceral endoderm. F9 cells were incubated with or without RA at 0, 24, 48, and 72 h. Total proteins extracted were separated by two‐dimensional electrophoresis (2‐DE) and the protein patterns on the gels were comparatively analyzed by computer. Approximately 1,100 protein spots were detected in the F9 proteome, within the pH 3–10 range. Fourteen protein spots which the levels of expression were found to be altered dramatically during the F9 cells differentiating, and were identified by MALDI‐TOF MS or ESI‐MS/MS. These proteins included metabolism enzymes, HSP60s, RAN, hnRNP K, FUBP1, VDAC1, STI1, and prohibitin. These proteins are involved in cellar metabolism, gene expression regulation, stress response, and apoptosis, respectively. The data from proteomic analyze are consistent with the result obtained from Western blot analysis. This study increases our understanding of the proteomics changes during F9 cells differentiation induced by RA. J. Cell. Biochem. 113: 1811–1819, 2012. © 2012 Wiley Periodicals, Inc.     

Comparative proteomic analysis reveals differential expression of Hsp25 following the directed differentiation of mouse embryonic stem cells

Murine embryonic stem (ES) cells can be committed to neural differentiation with high efficiency in culture through the use of feeder- and serum-free media. This system is proving to be an excellent model to study processes involved in ES cell commitment to neural cell fate. We used this approach to generate neurogenic embryoid bodies (NEBs) in a serum-free culture system to perform proteomic analysis of soluble fractions and identify early changes in protein expression as ES cells differentiate. Ten candidate proteins were altered significantly in expression levels. One of the most significant alterations was for the small heat shock protein Hsp25. Three species of Hsp25 are detected in ES cells, and this expression pattern changes during the first 24 h of differentiation until expression is decreased to levels that are barely detectable at 4 days following differentiation. We used immunofluorescence studies to confirm that following ES cell differentiation, expression of Hsp25 becomes excluded from neural precursors as well as other differentiating cells, making it a potentially useful marker of early ES cell differentiation.     

Synthetically modified methoxsalen for enhanced cytotoxicity in light and dark reactions

Linear furocoumarins, also known as psoralens, are clinically useful photo-activated pharmaceuticals employed to address hyperproliferative skin diseases. Seven diverse cytotoxic pharmacophores have been synthetically attached to 8-methoxypsoralen via a 5-amino functionality. The resulting unique set of compounds was evaluated for dark and light toxicity against PAM212 keratinocytes in culture.     

Expression of high molecular weight (67K) keratin in human keratinocytes cultured on dead de-epidermized dermis

The influence of living dermal tissue upon epidermal differentiation during embryonic development as well as in vitro culture has been documented. Living dermal tissue contains both cellular and matricial elements. In the present study, third-passage subcultured adult human keratinocytes were either seeded on plastic dishes or recombined with dead de-epidermized dermis and further cultured for 3 weeks. After this time, keratins were extracted and analysed by one- and two-dimensional gel electrophoresis. The 67K keratin subunit, which is thought to be involved in the process of in vivo type skin differentiation, was absent in ordinary cultures; however, it was expressed in air-exposed cultures on dead de-epidermized dermis. Quantitatively, however, it did not reach the in vivo level. This suggests that in principle, the induction of the expression of this protein does not require the presence of living dermal cells.     

Developmental regulation of peptidylglycine alpha-amidating monooxygenase (PAM) in rat heart atrium and ventricle. Tissue-specific changes in distribution of PAM activity, mRNA levels, and protein forms

The high levels of peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) found in adult rat atrium led us to examine PAM expression in rat atrium and ventricle from embryonic day 14 through adulthood. Immunocytochemical studies using antisera to PAM identified cardiocytes as the major site of PAM expression in atrium and ventricle throughout development. Levels of PAM mRNA and PAM activity exhibited distinctly different developmental profiles in atrium and ventricle. Ventricular PAM mRNA and PAM activity were highest from embryonic days 14 through 18, declined at the time of birth, rose slightly during the first postnatal week, and declined toward adult levels. Atrial PAM mRNA and PAM activity were low at embryonic day 14, rose to a peak immediately before birth, declined at the time of birth, and then rose after birth. Levels of atrial PAM mRNA and PAM activity were not directly correlated at all developmental stages. Two major forms of PAM mRNA (4.2 +/- 0.1 and 3.8 +/- 0.1 kilobase(s] were identified in atrium and ventricle throughout development. The prevalence of the two forms varied with developmental stage, with atrium and ventricle containing similar forms at each stage. Western blots of atrial and ventricular membranes revealed the existence of a developmental stage-specific distribution of PAM protein among forms ranging in mass from 125 to 94 kDa. In both atrium and ventricle PAM activity was primarily soluble from embryonic days 14 through 16 and primarily particulate after birth. The role of PAM in the heart is not yet clear, but the presence of tissue-specific and developmentally regulated alterations in PAM mRNA, PAM protein, and PAM activity suggests that this peptide processing enzyme plays a key role in the heart.     

Involvement of cell surface HSP90 in cell migration reveals a novel role in the developing nervous system

Heat shock protein HSP90 plays important roles in cellular regulation, primarily as a chaperone for a number of key intracellular proteins. We report here that the two HSP90 isoforms, alpha and beta, also localize on the surface of cells in the nervous system and are involved in their migration. A 94-kDa surface antigen, the 4C5 antigen, which was previously shown to be involved in migration processes during development of the nervous system, is shown to be identical to HSP90alpha using mass spectrometry analysis. This identity is further confirmed by immunoprecipitation experiments and by induction of 4C5 antigen expression in heat shock-treated embryonic rat brain cultures. Moreover, immunocytochemistry on live cerebellar rat cells reveals cell surface localization of both HSP90alpha and -beta. Cell migration from cerebellar and sciatic nerve explants is inhibited by anti-HSP90alpha and anti-HSP90beta antibodies, similarly to the inhibition observed with monoclonal antibody 4C5. Moreover, immunostaining with rhodamine-phalloidin of migrating Schwann cells cultured in the presence of antibodies against both alpha and beta isoforms of HSP90 reveals that HSP90 activity is associated with actin cytoskeletal organization, necessary for lamellipodia formation.