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Abstract Background Therapeutically bioactive cell populations are currently understood to promote regenerative outcomes in vivo by leveraging mechanisms of action including secretion of growth factors, site specific engraftment and directed differentiation. Constitutive cellular populations undoubtedly participate in the regenerative process. Adipose tissue represents a source of therapeutically bioactive cell populations. The potential of these cells to participate in various aspects of the regenerative process has been demonstrated broadly. However, organ association of secretory and developmental markers to specific peri-organ adipose depots has not been investigated. To characterize this topographical association, we explored the potential of cells isolated from the stromal vascular fraction (SVF) of kidney sourced adipose to express key renal associated factors. Results We report that renal adipose tissue is a novel reservoir for EPO expressing cells. Kidney sourced adipose stromal cells demonstrate hypoxia regulated expression of EPO and VEGF transcripts. Using iso-electric focusing, we demonstrate that kidney and non-kidney sourced adipose stromal cells present unique patterns of EPO post-translational modification, consistent with the idea that renal and non-renal sources are functionally distinct adipose depots. In addition, kidney sourced adipose stromal cells specifically express the key renal developmental transcription factor WT1. Conclusions Taken together, these data are consistent with the notion that kidney sourced adipose stromal (KiSAS) cells may be primed to recreate a regenerative micro-environment within the kidney. These findings open the possibility of isolating solid-organ associated adipose derived cell populations for therapeutic applications in organ-specific regenerative medicine products.

Abstract Background The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, α, γ1 and δ, bind to pRb and dephosphorylate its Ser/Thr sites in a sequential and site-specific way. The pRb-C terminal has been reported to be necessary and sufficient for PP1α binding. The present study investigated whether the three PP1 isoforms from mitotic or asynchronous HeLa cells associate differentially with wild-type and pRb mutants, as well as the holoenzyme composition of the pRb-directed PP1. Results The requirement for the entire pRb molecule to achieve optimal PP1-binding was indicated by the fact that full-length pRb displayed the highest affinity for all three PP1 isoforms. Ser/Thr-to-Ala substitution for up to 14 pRb sites did not affect the ability of pRb to bind the PP1 isoforms derived from mitotic or asynchronous HeLa cells, thus suggesting that the phosphate-accepting residues on pRb do not regulate the interaction with PP1. To probe for the presence of PP1 targeting subunits in the pRb-directed PP1 complex, PP1 from mitotic or asynchronous HeLa cells was isolated by affinity chromatography on GST-Rb (either full-length or its deletion mutants Rb-big pocket or Rb-C-terminal). The PP1 was always obtained as free catalytic subunit, displaying all three isoforms, thus suggesting direct interaction between pRb and PP1. The direct association was confirmed by the ability of pRb to pull-down purified PP1 catalytic subunits and by in vitro reconstitution of a complex between PP1 catalytic subunit and the pRb-C-terminal. Conclusion The work indicated that the full length of the pRb molecule is required for optimal interaction with the PP1 isoforms and that the association between pRb and PP1 isoforms is direct.

Abstract Background One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1). Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression. One physiological substrate for PP1 appears to be the product of the retinoblastoma susceptibility gene (pRB), a demonstrated tumor suppressor. The growth suppressive activity of pRB is regulated by its phosphorylation state. Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation. As a first step towards addressing this question, we developed an inducible PP1 expression system to investigate the regulation of PP1 activity. Results We have established a cell line for inducing protein expression of the type 1, alpha-isotype, serine/threonine protein phosphatase (PP1α). A plasmid encoding a fusion protein of the catalytic subunit of PP1α with a 6-histidine peptide (6His) and a peptide from hemagluttinin (HA) was transfected into the UMUC3 transitional cell carcinoma cell line, previously transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo. A stable cell line designated LLWO2F was established by selection with hygromycin B. 6His-HA-PP1α protein appeared in cell lysates within two hours following addition of doxycycline to the culture medium. This protein localizes to the nucleus as does endogenous PP1α, and was shown to associate with PNUTS, a PP1-nuclear targeting subunit. Like endogenous PP1α, immunocomplexed 6His-HA-PP1α is active toward phosphorylase a and the product of the retinoblastoma susceptibility gene, pRB. When forcibly overexpressing 6His-HA-PP1α, there is a concomitant decrease in endogenous PP1α levels. Conclusions These data suggest the existence of an autoregulatory mechanism by which PP1α protein levels and activity remain relatively constant. RT-PCR analyses of isolated polysome fractions support the notion that this putative autoregulatory mechanism is exerted, at least in part, at the translational level. Implications of these findings for the study of PP1α function in vivo are discussed.

Studies of families with Birt-Hogg-Dubé syndrome (BHD) have recently revealed protein-truncating mutations in the BHD gene, leading to tumorigenesis of the skin and of different cell types of kidney. To additionally evaluate the role of BHD in kidney tumorigenesis, we studied 39 sporadic renal tumors of different cell types: 7 renal oncocytomas, 9 chromophobe renal cell carcinomas (RCCs), 11 papillary RCCs, and 12 clear cell RCCs. We screened for BHD mutations and identified a novel somatic mutation in exon 13: c.1939_1966delinsT in a papillary RCC. We performed loss of heterozygosity (LOH) analysis on 28 matched normal/tumor sets, of which 10 of 28 (36%) demonstrated LOH: 2 of 6 (33%) chromophobe RCCs, 5 of 6 (83%) papillary RCCs, 3 of 12 (25%) clear cell RCCs, but 0 of 4 renal oncocytomas. BHD promoter methylation status was examined by a methylation-specific PCR assay of all of the tumors. Methylation was detected in 11 of 39 (28%) sporadic renal tumors: 2 of 7 (29%) renal oncocytomas, 1 of 9 (11%) chromophobe RCCs, 4 of 11 (36%) papillary RCCs, and 4 of 12 (33%) clear cell RCCs. Five tumors with methylation also exhibited LOH. Mutation and methylation were absent in 9 kidney cancer cell lines. Our results showed that somatic BHD mutations are rare in sporadic renal tumors. The alternatives, LOH and BHD promoter methylation, are the two possible inactivating mechanisms involved. In conclusion, unlike other hereditary kidney cancer-related genes (i.e., VHL and MET), which are cell type-specific, BHD is involved in the entire spectrum of histological types of renal tumors, suggesting its major role in kidney cancer tumorigenesis.