Hsp72 (HSPA1A) Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment

Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in β-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP) which forms toxic aggregates that destroy pancreatic β-cells. Heat shock proteins (HSP) play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A) prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic β-cells. Moreover, Hsp72 (HSPA1A) overexpression in pancreatic β-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A) prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP) that is implicated in amyloid formation and β-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A), significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A) as a potential treatment to prevent β-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity

A mouse model for triple-negative breast cancer tumor-initiating cells (TNBC-TICs) exhibits similar aggressive phenotype to the human disease

<p>Abstract</p> <p>Background</p> <p>Triple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems.</p> <p>Methods</p> <p>To understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis.</p> <p>Results</p> <p>We isolated tumor-initiating cells (TICs) by sorting for CD24⁺/CD44^high/ALDH1⁺cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24⁺/CD44^high/ALDH1⁺cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24^-/CD44^-/ALDH1^-cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A).</p> <p>Conclusions</p> <p>Taken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC.</p

Genome fingerprinting of the silkworm, Bombyx mori, using random arbitrary primers

The random amplified polymorphic DNA (RAPD) technique was used to study DNA profiling of thirteen silkworm genotypes. The genotypes included six diapausing and seven nondiapausing varieties that represent a high degree of divergence with respect to geographic origin, and morphological, qualitative, quantitative and biochemical characters. Two hundred sixteen amplified products were generated using 40 random primers. Genotype-specific amplification products were identified. Amplification products specific to diapausing genotypes were also identified. Segregation of the RAPD marker was analyzed in a backcross population and found to be inherited as dominant Mendelian traits. Based on pairwise comparison of amplified products, the genetic similarity coefficient was calculated using the NeiLi similarity coefficient, and cluster analysis was performed by a hierarchical clustering technique. Silkworm genotypes were clustered into two groups, one consisting of six diapausing and the other of seven nondiapausing genotypes. The results of our study suggest that the RAPD technique could be used as a powerful tool to generate genetic markers that are linked to traits of interest in the silkworm

Hsp72 expression improves the solubility of h-proIAPP aggregates.

<p><b>A.</b> Transgenic YFP + Hsp72 <i>C</i>. <i>elegans</i> model was generated by co-injection of 20 ng/μl of plasmid pPR18 (that expresses YFP in muscles) together with 30 ng/μl of Hsp72 expressed in the same tissue (plasmid pPR21) to serve as a soluble control (top panels). <i>C</i>. <i>elegans</i> injected with 20 ng/μl of h-proIAPP tagged with YFP expressed in muscles (plasmid pPR3) was used as a control for protein aggregation (middle panels). Transgenic h-proIAPP::YFP + Hsp72 <i>C</i>. <i>elegans</i> animals were generated by gonad co-injection of 20 ng/μl of plasmid pPR3 and 30 ng/μl of plasmid pPR21 (bottom panels). Transgenic animals were subjected to FRAP analysis (square). Data was collected before photobleaching (left panels), 10 seconds after photobleaching (middle panels) and 250 seconds after photobleaching (right panels). Images were obtained using an inverted confocal microscope. Results are representative of one experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm. <b>B.</b> Data represents the quantification of relative fluorescence intensity, RFI ± SEM during recovery after photobleaching of transgenic h-proIAPP::YFP <i>C</i>. <i>elegans</i> model (filled circles), transgenic h-proIAPP::YFP + Hsp72 <i>C</i>. <i>elegans</i> animals (open circles), and transgenic YFP + Hsp72 <i>C</i>. <i>elegans</i> model (filled triangles). Data are the mean of at least three independently performed experiments. *p<0.05 versus h-proIAPP::YFP, Error Bar = SEM.</p

Expression of h-proIAPP in <i>C</i>. <i>elegans</i> results in protein insolubility and aggregation.

<p>Transgenic h-proIAPP <i>C</i>. <i>elegans</i> model was generated by gonad microinjection of 20 ng/μl of plasmids pPR3, pPR4 and pPR5. Transgenic m-proIAPP <i>C</i>. <i>elegans</i> model was generated by gonad microinjection of 20 ng/μl of plasmids pPR8, pPR9 and pPR10. <b>A.</b> h-proIAPP and m-proIAPP tagged with YFP were expressed in body wall muscles, vulva muscles and anal depressor muscles under <i>lev-11</i> promoter; in pharynx under <i>tnt-4</i> promoter, and in neurons under <i>aex-3</i> promoter. Neuronal expression of proIAPP secreted into the body cavity was observed in the coelomocytes of the h-proIAPP model and distributed in the intestinal region in both h-proIAPP and m-proIAPP <i>C</i>. <i>elegans</i> models. Images were created using maximum intensity projections on all the planes of the acquired stacks except coelomocytes where images were taken in a single plane for better visualization. Arrows indicate areas of fluorescence. DIC images were taken to visualize structures. Results are a representative experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm. <b>B.</b> Data represent the mean fluorescence intensity (MFI) ± SD measured using a sum intensity projection on all the planes of the acquired stacks of m-proIAPP (open bars) and h-proIAPP (filled bars) tagged with YFP expressed in body wall muscles, vulva muscles, anal depressor muscles and pharynx. *p<0.05; **p<0.01 versus m-proIAPP. <b>C.</b> Transgenic h-proIAPP <i>C</i>. <i>elegans</i> tagged with YFP were subjected to FRAP analysis (square). Data are from vulva muscles (row 1), anal depressor (row 2), pharynx (row 3) and coelomocytes (row 4) before FRAP (left panels), 10 seconds after FRAP (middle panels) 250 seconds after FRAP (right panels). Images were obtained using an inverted confocal microscope. Results are representative experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm.</p

Hsp72 reduces endogenous h-IAPP toxicity in pancreatic β-cells.

<p><b>A.</b> Beta-TC-6 cells were transfected and cell viability was assessed by MTS assay. Data represent percentage of live cells ± SD (filled bars) when transfected with vector pCMV6-XL5 (control) or human Hsp72::EGFP cDNA clone or m-proIAPP cDNA clone or h-proIAPP cDNA clone or both h-proIAPP and human Hsp72::EGFP vectors. Data represents the sum of three independently performed experiments. **p<0.01 versus respective controls, n = 3. <b>B.</b> At the end of the transfection period, phase contrast and fluorescence images were obtained. Data is a representative experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm. <b>C.</b> Beta-TC-6 cells were transfected with empty vector pCMV6-XL5 (control), or m-proIAPP cDNA clone or h-proIAPP cDNA clone or with both h-proIAPP and human Hsp72::EGFP vectors. After transfection, samples were collected and examined for the expression of h-IAPP by Western blot analysis. Data is a representative experiment from at least three independently performed experiments with similar results. <b>D.</b> Beta-TC-6 cells were transfected with empty vector pCMV6-XL5 (control), or human Hsp72::EGFP cDNA clone, or with both h-proIAPP and human Hsp72::EGFP vectors. After transfection, samples were collected and examined for the expression of Hsp72 by Western blot analysis. Data is a representative experiment from at least three independently performed experiments with similar results.</p

Construction and identification of human and mouse proIAPP <i>C</i>. <i>elegans</i> vectors.

<p><b>A.</b> Schematic representation of human-proIAPP and mouse-proIAPP tissue-specific vector constructs. cDNA of human and mouse proIAPP with human and mouse signal peptide, respectively, were cloned into the BamH1 restriction site of the pSX95.77YFP <i>C</i>.<i>elegans</i> vector to generate human and mouse pSX95.77YFP-proIAPP transgenes. Resulting constructs were digested with Sal1, blunt-ended and ligated into Gateway Cassette C.1 upstream of human and mouse coding sequences to create vectors pNG1 and pNG2. Destination vectors pNG1 and pNG2 were LR recombined with pLR22, pLR25 and pLR35 entry clones to generate human proIAPP plasmids: pPR3, pPR4 and pPR5 and mouse proIAPP plasmids: pPR8, pPR9 and pPR10. <b>B.</b> h-proIAPP sequence of construct pSX95.77YFP-prohIAPP with human signal peptide. Preproh-IAPP (1–89) peptide amino acid sequence is shown in black italics. Signal peptide sequence is shown in red. <b>C.</b> m-proIAPP sequence present in construct pSX95.77YFP-promIAPP with mouse signal peptide. Preprom-IAPP peptide amino acid sequence is shown in black italics. Signal peptide sequence is shown in red. Restriction sites used in the generation of these plasmids are underlined. No stop codon was included after each proIAPP sequence.</p

Expression of h-proIAPP in <i>C</i>. <i>elegans</i> correlates with growth retardation phenotype.

<p>Developmental phenotype was studied in transgenic h-proIAPP <i>C</i>. <i>elegans</i> model, transgenic m-proIAPP <i>C</i>. <i>elegans</i> model and pBX1 control animals by quantifying the number of animals in the larvae 1 (L1) to larvae 3 (L3) stages (open bars) versus larvae 4 (L4) and adult stages (filled bars) seventy-two hours after ten to fifteen gravid hermaphrodites were treated with bleach to release eggs. Data represent the percentage of animals found at larvae 1 to larvae 3 stages and the percentage of animals found at larvae 4 and adult stages ± S.D. p<0.05 versus respective control, n = 3.</p