CBL407 Sigma-AldrichAnti-PCNA Antibody, clone PC10

Lizard skin can produce scales during embryonic development, tail regeneration, and wound healing; however, underlying molecular signaling and extracellular matrix protein expression remains unknown. We mapped cell proliferation, signaling and extracellular matrix proteins in regenerating and developing lizard scales in different body regions with different wound severity. Following lizard tail autotomy (self-amputation), de novo scales regenerate from regenerating tail blastema. Despite topological differences between embryonic and adult scale formation, asymmetric cell proliferation produces the newly formed outer scale surface. Regionally different responses to wounding were observed; open wounds induced better scale regeneration from tail skin than trunk skin. Molecular studies suggest NCAM enriched dermal regions exhibit higher cell proliferation associated with scale growth. β-catenin may be involved in epidermal scale differentiation. Dynamic tenascin-C expression suggests its involvement in regeneration. We conclude that different skin regions exhibit different competence for de novo scale formation. While cellular and morphogenetic paths differ during development and regeneration of lizard scale formation, they share general proliferation patterns, epithelial-mesenchymal interactions and similar molecular modules composed of adhesion and extracellular matrix molecules.

Infection by the autonomous parvovirus minute virus of mice (MVM) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication. Although p53 remains activated, p21 protein levels remain low throughout the course of infection. We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers. PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA. PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not. Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA.

Feathers are hallmark avian integument appendages, although they were also present on theropods. They are composed of flexible corneous materials made of α- and β-keratins, but their genomic organization and their functional roles in feathers have not been well studied. First, we made an exhaustive search of α- and β-keratin genes in the new chicken genome assembly (Galgal4). Then, using transcriptomic analysis, we studied α- and β-keratin gene expression patterns in five types of feather epidermis. The expression patterns of β-keratin genes were different in different feather types, whereas those of α-keratin genes were less variable. In addition, we obtained extensive α- and β-keratin mRNA in situ hybridization data, showing that α-keratins and β-keratins are preferentially expressed in different parts of the feather components. Together, our data suggest that feather morphological and structural diversity can largely be attributed to differential combinations of α- and β-keratin genes in different intrafeather regions and/or feather types from different body parts. The expression profiles provide new insights into the evolutionary origin and diversification of feathers. Finally, functional analysis using mutant chicken keratin forms based on those found in the human α-keratin mutation database led to abnormal phenotypes. This demonstrates that the chicken can be a convenient model for studying the molecular biology of human keratin-based diseases.

B-lymphocyte-induced nuclear maturation protein 1 (BLIMP1) was previously reported to define a sebaceous gland (SG) progenitor population in the epidermis. However, the recent identification of multiple stem cell populations in the hair follicle junctional zone has led us to re-evaluate its function. We show, in agreement with previous studies, that BLIMP1 is expressed by postmitotic, terminally differentiated epidermal cells within the SG, interfollicular epidermis, and hair follicle. Epidermal overexpression of c-Myc results in loss of BLIMP1(+) cells, an effect modulated by androgen signaling. Epidermal-specific deletion of Blimp1 causes multiple differentiation defects in the epidermis in addition to SG enlargement. In culture, BLIMP1(+) sebocytes have no greater clonogenic potential than BLIMP1(-) sebocytes. Finally, lineage-tracing experiments reveal that, under steady-state conditions, BLIMP1-expressing cells do not divide. Thus, rather than defining a sebocyte progenitor population, BLIMP1 functions in terminally differentiated cells to maintain homeostasis in multiple epidermal compartments.

Histone deacetylases (HDACs) are present in the epidermal layer of the skin, outer root sheath, and hair matrix. To investigate how histone acetylation affects skin morphogenesis and homeostasis, mice were generated with a K14 promoter-mediated reduction of Hdac1 or Hdac2. The skin of HDAC1 null (K14-Cre Hdac1(cKO/cKO)) mice exhibited a spectrum of lesions, including irregularly thickened interfollicular epidermis, alopecia, hair follicle dystrophy, claw dystrophy, and abnormal pigmentation. Hairs are sparse, short, and intermittently coiled. The distinct pelage hair types are lost. During the first hair cycle, hairs are lost and replaced by dystrophic hair follicles with dilated infundibulae. The dystrophic hair follicle epithelium is stratified and is positive for K14, involucrin, and TRP63, but negative for keratin 10. Some dystrophic follicles are K15 positive, but mature hair fiber keratins are absent. The digits form extra hyperpigmented claws on the lateral sides. Hyperpigmentation is observed in the interfollicular epithelium, the tail, and the feet. Hdac1 and Hdac2 dual transgenic mice (K14-Cre Hdac1(cKO/cKO) Hdac2(+/cKO)) have similar but more obvious abnormalities. These results show that suppression of epidermal HDAC activity leads to improper ectodermal organ morphogenesis and disrupted hair follicle regeneration and homeostasis, as well as indirect effects on pigmentation.

The formation of the bipolar spindle is responsible for accurate chromosomal segregation during mitosis. The dynamic instability of microtubules has an important role in this process, and has been shown to be an effective target for cancer chemotherapy. Several agents that target non-microtubule mitotic proteins, including the motor protein Eg5, Aurora kinases and Polo-like kinases, are currently being developed as chemotherapeutic drugs. However, because the efficacies of these drugs remain elusive, new molecular targets that have essential roles in tumor cells are desired. Here, we provide in vivo evidence that transforming acidic coiled-coil-3 (Tacc3) is a potential target for cancer chemotherapy. Using MRI, we showed that Tacc3 loss led to the regression of mouse thymic lymphoma in vivo, which was accompanied by massive apoptosis. By contrast, normal tissues, including the thymus, showed no overt abnormalities, despite high Tacc3 expression. in vitro analysis indicated that Tacc3 depletion induced multi-polar spindle formation, which led to mitotic arrest, followed by apoptosis. Similar responses have been observed in Burkitt's lymphoma and T-ALL. These results show that Tacc3 is a vulnerable component of the spindle assembly in lymphoma cells and is a promising cancer chemotherapy target.

Feathers have complex forms and are an excellent model to study the development and evolution of morphologies. Existing chicken feather mutants are especially useful for identifying genetic determinants of feather formation. This study focused on the gene F, underlying the frizzle feather trait that has a characteristic curled feather rachis and barbs in domestic chickens. Our developmental biology studies identified defects in feather medulla formation, and physical studies revealed that the frizzle feather curls in a stepwise manner. The frizzle gene is transmitted in an autosomal incomplete dominant mode. A whole-genome linkage scan of five pedigrees with 2678 SNPs revealed association of the frizzle locus with a keratin gene-enriched region within the linkage group E22C19W28_E50C23. Sequence analyses of the keratin gene cluster identified a 69 bp in-frame deletion in a conserved region of KRT75, an α-keratin gene. Retroviral-mediated expression of the mutated F cDNA in the wild-type rectrix qualitatively changed the bending of the rachis with some features of frizzle feathers including irregular kinks, severe bending near their distal ends, and substantially higher variations among samples in comparison to normal feathers. These results confirmed KRT75 as the F gene. This study demonstrates the potential of our approach for identifying genetic determinants of feather forms.

Chronic kidney disease is a leading cause of death in the United States. Tubulointerstitial fibrosis (TIF) is considered the final common pathway leading to end-stage renal disease (ESRD). Here, we used pharmacologic, genetic, in vivo, and in vitro experiments to show that activation of the Notch pathway in tubular epithelial cells (TECs) in patients and in mouse models of TIF plays a role in TIF development. Expression of Notch in renal TECs was found to be both necessary and sufficient for TIF development. Genetic deletion of the Notch pathway in TECs reduced renal fibrosis. Consistent with this, TEC-specific expression of active Notch1 caused rapid development of TIF. Pharmacologic inhibition of Notch activation using a γ-secretase inhibitor ameliorated TIF. In summary, our experiments establish that epithelial injury and Notch signaling play key roles in fibrosis development and indicate that Notch blockade may be a therapeutic strategy to reduce fibrosis and ESRD development.

Monoclonal antibodies (MAb) to a 36 KD protein, proliferating cell nuclear antigen (PCNA/cyclin), have been previously shown to be capable of identifying proliferating cells in vitro as well as in alcohol-fixed, paraffin-embedded tissue specimens. The routine use of these anti-PCNA/cyclin MAb in investigative studies and in diagnostic pathology requires a clearer understanding of the distribution of PCNA/cyclin in the different cell populations found in tissue specimens. We therefore compared the ability of MAb to three nucleus-associated proliferation markers (MAb 19A2 to PCNA/cyclin; Ki-67 to an undefined proliferation-related marker; BU-1 to 5'-bromodeoxyuridine (BrdU) incorporated into DNA) to identify the proliferating cell fraction of various cells in vitro. The cell lines were chosen to represent a spectrum of proliferation rates (high to low) and cell lineage (mesenchymal vs epithelial, non-transformed vs malignant): (a) HeLa and A-431 (two malignant carcinoma cell lines with high proliferation rates); (b) SK-5 (a non-transformed fibroblast cell line with a low proliferation rate); (c) HUVE (a non-transformed human umbilical vein endothelial cell line with a low proliferation rate). Single and double labeling immunofluorescence studies were performed after uniform 1-hr incubations with BrdU. Comparison of the overlapping distributions of detectable PCNA/cyclin expression and BrdU incorporation demonstrated substantial qualitative and quantitative differences between the different cell lines. In two of the four cell lines (HeLa, A-431) the BrdU staining distributions formed inclusive subsets of the PCNA-positive cell populations. In the HUVE cell line the two populations overlapped incompletely. In one cell line, SK-5, the two populations were mutually exclusive. MAb Ki-67 demonstrated a pattern in the SK-5 cell line that was strongly predictive of PCNA positivity, while showing no associated patterns in the other three cell lines. We conclude that PCNA/cyclin expression detected by MAb may define different cell subpopulations in different cell types relative to those incorporating BrdU or expressing the target antigen for Ki-67. This has implications for the clinical study of mixed cell populations using these antibodies.

The proliferating cell nuclear antigen, PCNA, has recently been identified as the polymerase delta accessory protein. PCNA is essential for cellular DNA synthesis and is also required for the in vitro replication of simian virus 40 (SV40) DNA where it acts to coordinate leading and lagging strand synthesis at the replication fork. The cDNA for rat PCNA was cloned into a series of bacterial expression vectors and the resulting protein used to immunize mice. Eleven new monoclonal antibodies to PCNA have been isolated and characterized. Some of the antibodies recognize epitopes conserved from man to fission yeast. Immunocytochemical analysis of primate epithelial cell lines showed that the antibodies recognized antigenically distinct forms of PCNA and that these forms were localized to different compartments of the nucleus. One antibody reacted exclusively with PCNA in the nucleolus. These results suggest that the PCNA protein may fulfil several separate roles in the cell nucleus associated with changes in its antigenic structure.